Lettuce plants comprising resistance against Nasonovia ribisnigri biotype 1

ABSTRACT

The present invention relates to the field of lettuce breeding, in particular to Quantitative Trait Loci for resistance against the lettuce aphid Nasonovia ribisnigri biotype Nr:1 and to cultivated lettuce comprising one or more of these Quantitative Trait Loci.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 National Phase of PCT ApplicationNo. PCT/EP2015/075127 filed Oct. 29, 2015, which claims benefit toEuropean Patent Application No. 14191135.4 filed Oct. 30, 2014, thedisclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to cultivated lettuce (Lactuca sativa)seeds, plants and plant parts comprising one or more introgressionfragments from a wild lettuce, such as Lactuca virosa, on chromosome 6and/or chromosome 7, whereby the introgression fragment comprises aQuantitative Trait Locus (QTL) for resistance against Nasonoviaribisnigri biotype 1 (also called herein Nr:1 or biotype Nr:1), referredto as QTL6.1 (for the QTL on chromosome 6) and QTL7.1 and 7.2 (for twoQTLs on chromosome 7). The present invention also relates to cultivatedlettuce (Lactuca sativa) seeds, plants and plant parts grown from theseeds, that are resistant to Nasonovia ribisnigri biotype 1 due to thepresence of an introgression fragment from L. virosa comprising QTL6.1and/or QTL7.1 (and/or QTL7.2) as well as to progenies of the plants andpropagation material for producing the plants. The invention furtherrelates to wild lettuce sources of the resistance-conferring QTLs foruse in breeding Nr:1 resistant lettuce plants.

BACKGROUND OF THE INVENTION

The lettuce aphid (Nasonovia ribisnigri (Mosley)) is a major pestoccurring in lettuce worldwide. The problem started to be severe forlettuce production in the 1970's in North Western Europe and spreadrapidly all across Europe. Then, in the 1980's, the aphid was detectedin Canada. Later on, the problem was reported in the USA (California andArizona). More recently, the lettuce aphid was found in New Zealand andAustralia.

Lettuce aphids can colonize lettuce plants at any plant stage and feedpreferably from younger leaves. Large amount of aphids on the plant areable to reduce plant growth and deform the shape of the head so that thelettuce heads are then not marketable. The presence of high amounts ofaphids in lettuce heads is a reason for retailers to refuse to buylettuce from growers. At young plant stage it is possible to control thelettuce aphid using insecticide. Several products were reported to beefficient in controlling aphid populations. However, resistance tochemicals were reported in some aphid populations. Moreover, at olderdevelopmental stages it is not possible to control aphids usinginsecticides, as chemical products cannot enter into the lettuce head.

Since 2007, two biotypes of lettuce aphid have been known in Europe,which were designated biotype Nr:0 and Nr:1. Complete and partialresistance against Nasonovia ribisnigri biotype Nr:0 were found inLactuca virosa, a wild relative of lettuce (Eenink and Dieleman,Euphytica 32(3), 691-695 (1982)). The complete resistance was due to asingle dominant gene, termed the Nr gene. The Nr gene was transferredfrom L. virosa accession IVT280 into cultivated L. sativa and was highlyeffective (Arend et al. 1999, Eucarpia Leafy Vegetables '99. PalackyUniversity, Olomouc, Czech Republic, p 149-157).

However, breeders experienced that the release of varieties resistant tolettuce aphid was not straightforward. The Nr resistance gene was foundtightly linked to recessive genes conferring strong negativeside-effects. Plants homozygous for the Nr gene showed a reduced growth,a lighter green colour and accelerated degradation of chlorophyll in theolder leaves. This negative phenotype was also referred to as the“Compact Growth and Rapid Ageing” phenotype or “CRA phenotype” and itwas possible to find recombinant lettuce plants in which the Nr gene waspresent in homozygous form, but in which the CRA phenotype was notexpressed (see, e.g., EP 0921720 B1). These recombinant plants, in whicha recombination event (i.e. meiotic crossing-over) had taken placebetween the Nr gene and the linked recessive genes, served as the sourceof the Nr resistance gene that was not linked to the negativeside-effect phenotype.

The Nr resistance gene from IVT280 (CGN04683) is widely used incommercial lettuce cultivars, such as cultivars ‘Barcelona’, ‘Mafalda’(both Nunhems B.V.) and many others.

Other sources of biotype Nr:0 resistance are still being sought, as thewide-scale use of a single resistance gene faces the threat ofresistance breakdown. Genes which have different resistance mechanismscan be effectively employed in such circumstances. For example newbiotype Nr:0 resistance genes were found in an L. serriola accessions PI491093 and in an L. virosa accession PI 274378 (Mc Creight 2008HortScience 43:1355-1358; McCreight and Liu (2012), HortScience47(2):179-184). The resistance found in PI274378 was found to becomplete and allelic to the Nr gene from IVT280. In PI49093 partialresistance was found, and the authors proposed to designate thisresistance allele Nr0^(P) (in contrast to Nr0^(C) for the completeresistance allele found in PI274378). They suggested using this partialresistance allele in areas where the complete resistance allele has notyet been widely employed in order to delay or prevent emergence of aphidbiotypes that overcome Nr resistance.

Also resistance against Nasonovia ribisnigri biotype Nr:1 is soughtafter. Different resistance genes and resistance mechanisms are alsodesired regarding Nr:1, to prolong the use of resistance genes. Whenlarge scale use is made of one resistance gene, which has a certainresistance mechanism, the chances are high that resistance will beovercome by the aphid population, as happened in 2007 for the Nr gene inEurope, when the new Nasonovia ribisnigri biotype Nr: 1 appeared. Thus,the current situation is that the aphid biotype Nr:0 can still becontrolled by the single Nr gene derived from the IVT L. virosaaccession (for example in the USA, where biotype Nr:1 does not yetoccur), but that this gene is ineffective against aphid biotype Nr:1found in Europe. The Nr: 1 biotype was first found only in centralEurope, but is now spreading to other areas and in 2010 has also beenfound in fields in Spain (Cid et al. 2012, Arthropod-Plant Interactions6: 655-669).

Several publications describe sources which are suggested to contain aresistance gene against lettuce aphid biotype Nr:1. For example threeaccessions (CGN13361, CGN16266, CGN16272) were described to be resistantagainst both aphid biotypes Nr:0 and Nr:1 (Anonymous, 4, Nov. 2008,IP.COM document 000176078) and were suggested to be used in breeding forcombined Nr:0 and Nr:1 resistant lettuce. In this article L. virosaaccessions CGN16272 and CGN16266 are said to be used in backcrossingprograms with cultivar Daguan (Syngenta) and cultivar Funly (Syngenta),respectively (both these cultivars lack Nasonovia resistance genes), andthe offspring are said to show a resistance similar to the resistance ofthe donor accessions. Also markers for the resistance gene of CGN16272are said to be developed from crosses of CGN16272 with cultivar CobhamGreen (Anonymous, 4, Nov. 2008, supra). These three accessions were alsoanalyzed in Cid et al. (2012, supra) and were found to comprise highNr:0 resistance but only partial biotype Nr:1 resistance.

Cid et al. (2012, supra) also identified three L. virosa accessions withsome resistance against both biotypes Nr:1 and Nr:0, namely CGN16274,CGN21399 and CGN05148. In this study, the authors aim was to findresistance against both lettuce aphid biotypes, Nr: 0 and Nr: 1, insingle wild Lactuca accession. However, aphids of biotype Nr:1 are stillable to feed and reproduce on these wild accessions, albeit to a lowerextent than on the susceptible controls (see FIG. 4).

WO2011/058192 reports L. serriola 10G.913571 as being resistant againstbiotype Nr:1, although no data are provided to substantiate this claimand no indication of the level of resistance and methods to determinethis is given.

WO2012/066008 and WO2012/065629 also report Nr: 1 resistance from an L.serriola accession to be transferred into a bulk seed sample designated10G.913569. Again, no data are provided and no indication on theresistance level and methods to determine resistance is given.

Thus, in the prior art, such as Anonymous 2008, above, and Cid et al.2012, above, only a few wild accessions are identified on which theamount of Nr: 1 aphids is reduced to some extent and no genetic basis isprovided.

There remains a need for identifying genes which can confer resistanceagainst biotype Nr:1 in order to develop cultivated lettuce comprisingNr:1 resistance. The instant inventors looked for accessions which werethought to be susceptible to aphids of biotype Nr:0, in order toidentify (new) resistance gene(s) against biotype Nr: 1 in theseaccessions. In addition, they also looked for the identification ofgenes which can confer both free-choice and non-choice resistance. Theyfound a L. virosa accession (of which a representative sample of seedswas deposited under NCIMB42086) comprising high levels of resistanceagainst biotype Nr:1, both under free choice and non-choice conditions,and decided to try to map the resistance, in order to identify how manyand which L. virosa genome regions are responsible for conferring Nr:1resistance.

When trying to map the resistance, the inventors encountered severeproblems in creating a population of plants useable for QTL mapping(i.e. a mapping population which consists of individuals that haveundergone chromosomal meiotic recombination between the L. sativa and L.virosa genomes). The reason is likely that the chromosomes of L. virosaand L. sativa, which are two different species, are quite different,leading to crossing barriers and infertility, as well as potentialproblems during meiosis and crossing over. No useable F2 populationscould be generated, and only after many crosses with various recurrentparents the inventors succeeded in generating large enough backcrossfamilies which could be used in mapping studies. These mappingpopulations were also not easy to analyze using molecular markers andphenotyping, i.e. it was quite surprising that the inventors managed togenerate a genetic map with SNP markers that are polymorphic between therecurrent parent and the L. virosa accession, and were also able to mapNasonovia Nr:1 resistance onto that map.

Surprisingly, in the initial QTL mapping study (using a BC1 population)they did not find a single gene, but three genomic regions (of whichonly two were later also found in a different backcross population) ontwo different chromosomes of L. virosa which contribute to the Nr:1resistance. Both controlled environment inoculations (free choice andnon-choice) and field data (also free choice and non-choice) showed highlevels of Nr:1 resistance, against three geographically distinct Nr:1biotypes (Germany, France and Spain). In fact, in field evaluations inSpain (Murcia) semi-adult and adult plants of the accession NCIMB42086did not have any Nr:1 aphids on their leaves in both free-choice andnon-choice tests.

In the later mapping study, two of the QTLs (QTL6.1 and QTL7.1) werefound again and the QTL region could be narrowed down. This secondmapping study does not invalidate the results of the first study, andall three QTLs are encompassed herein.

It is an object of the invention to provide three QTLs (designatedQTL6.1, QTL7.1 and/or QTL7.2) from L. virosa which can be used togenerate cultivated lettuce plants comprising resistance against biotypeNr:1.

It is also an object of the invention to provide cultivated lettuceplants comprising one or two or three QTLs (QTL6.1 and/or QTL7.1 and/orQTL7.2) introgressed from a wild lettuce, such as L. virosa, into the L.sativa genome, whereby the introgressions confer resistance againstbiotype Nr: 1.

Thus, different cultivated lettuce plants are encompassed herein: a)cultivated lettuce plants comprising only one QTL conferring Nr:1resistance, selected from QTL6.1, QTL7.1 and QTL7.2; b) cultivatedlettuce plants comprising two QTLs conferring Nr:1 resistance selectedfrom QTL6.1 and QTL7.1 and QTL7.2 (in one aspect a plant comprising bothQTL6.1 and QTL7.1 is a specific embodiment); c) cultivated lettuceplants comprising three QTLs conferring Nr:1 resistance selected fromQTL6.1, QTL7.1 and QTL7.2.

It is also an object of the invention to provide cultivated lettuceplants comprising one or two QTLs selected from QTL6.1 and QTL7.1introgressed from a wild lettuce, such as L. virosa, into the L. sativagenome, whereby the introgressions confer resistance against biotype Nr:1.

In one aspect the QTLs are obtainable from (are as in) seeds depositedunder accession number NCIMB42086. The introgression fragment(s)comprising the QTL(s) is/are detectable by a molecular marker assaywhich detects at least 1, 2, 3, 4, or more markers. In another aspectthe QTLs are obtainable from (are as in) other Nr:1 resistant wildlettuce accessions, especially in L. virosa accessions, whereby theintrogression fragment(s) is/are detectable by a molecular marker assaywhich detects at least 1, 2, 3, 4, or more (i.e. 5, 6, 7, 8, 9, 10, 11,12, or more) markers disclosed herein. In one aspect the L. virosaaccession is one of two types of accessions, and the introgressionfragment comprises a L. virosa accession specific SNP marker (named VSPfor Virosa Specific), selected from VSP1 and VSP2, both specific for oneL. virosa accession and VSP3 and VSP4, both specific for another L.virosa accession.

Despite problems in interspecific QTL mapping mentioned above, likeinfertility, segregation distortion, etc., the originally found QTLregions (which were originally mapped to a physical region spanning 60to 240 Mb on chromosome 6; and 170 to 235 Mb on chromosome 7 for QTL7.1;and 70 to 150 Mb for QTL 7.2) could be mapped to a region spanning 77 Mbto 161 Mb on chromosome 6 (comprising QTL6.1) and 203 Mb to 219 Mb onchromosome 7 comprising QTL7.1.

Thus, in one aspect a cultivated Lactuca sativa plant is providedcomprising an introgression fragment on chromosome 6 (comprising QTL6.1)and/or on chromosome 7 (comprising QTL7.1), each in homozygous orheterozygous form, wherein said introgression fragment confersresistance against Nasonovia ribisnigri biotype 1 (Nr:1). In one aspectthe introgression fragment comprises all or part of the region startingat 77 Mb on chromosome 6 and ending at 161 Mb on chromosome 6 and/or theintrogression fragment comprises all or part of the region starting at203 Mb on chromosome 7 and ending at 219 Mb on chromosome 7. See e.g.FIG. 3B, showing L. sativa chromosomes 6 and 7, where the gray barsillustrate the introgression fragments from a wild Nr:1 resistantaccession, such as a L. virosa accession (e.g. NCIMB42086) comprisingthe resistance conferring QTLs QTL6.1 and QTL7.1, or variants thereof.In one aspect an introgression fragment comprising QTL7.2 may alsooptionally be present in the cultivated lettuce plant.

It is understood that a smaller introgression fragment (i.e. comprisinga resistance conferring part of the above mentioned region spanning 77Mb to 161 Mb of chromosome 6) which retains the QTL6.1 (or variant) maybe a fragment having a size of 80 Mb, 70 Mb, 60 Mb, 50 Mb, 40 Mb, 30 Mb,20 Mb, 10 Mb, 5 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 100 kb, 50 kb or lessand comprise the QTL6.1 or a variant thereof. In one aspect the part isat least 5 kb, 10 kb, 20 kb in size, or more.

It is further understood that a smaller introgression fragment (i.e.comprising a resistance conferring part of the above mentioned regionspanning 203 Mb to 219 Mb of chromosome 7) which retains the QTL7.1 (orvariant) may be a fragment having a size of 15 Mb, 10 Mb, 5 Mb, 2.5 Mb,2 Mb, 1 Mb, 0.5 Mb, 100 kb, 50 kb or less and comprise the QTL7.1 or avariant thereof. In one aspect the part is at least 5 kb, 10 kb, 20 kbin size, or more.

In one aspect, the introgression fragment on chromosome 6 is detectableby a molecular marker assay which detects at least one, preferably atleast 2 or 3 or 4 or 5 (or more) of the markers selected from the groupconsisting of:

-   -   a) The CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP1.23 in SEQ ID NO: 23 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 23);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 2);    -   c) the TT or CT genotype for the Single Nucleotide Polymorphism        marker SNP2.24 in SEQ ID NO: 24 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 24);    -   the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 3);    -   d) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP1.23 and SNP_03 (e.g. in-between SNP1.23 and SNP2.24, SNP1.23        and SNP_02); or in between SNP_02 and SNP_03 (e.g. in-between        SNP_02 and SNP2.24); or in between SNP2.24 and SNP_03;    -   e) any wild lettuce genome specific marker especially L.        virosa-genome specific marker, located within a distance of 10        Mb, preferably within 5 Mb, of any marker selected from SNP1.23,        SNP_02, SNP2.24, or SNP_03.

Optionally, in one aspect, the introgression fragment comprises (and isdetectable by) a L. virosa accession specific marker selected from theGG or GT genotype for the Single Nucleotide Polymorphism marker VSP1 inSEQ ID NO: 26 (or in a sequence comprising substantial sequence identityto SEQ ID NO: 26) and the AA or AC genotype for the Single NucleotidePolymorphism marker VSP3 in SEQ ID NO: 27 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 27). Using the SNP markersVSP1 and VSP3 the introgression fragments comprising QTL6.1 from twodifferent L. virosa type accessions can be distinguished.

In another aspect, the introgression fragment at the far end ofchromosome 7 comprising QTL 7.1 (at a physical position between 203 Mband 219 Mb of chromosome 7) is detectable by a molecular marker assaywhich detects at least one, preferably at least 2 or 3 or 4 or 5 (ormore) of the markers selected from the group consisting of:

-   -   a. the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 17);    -   b. the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP17.25 in SEQ ID NO: 25 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 25);    -   c. the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18);    -   d. the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19);    -   e. any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_17 and SNP_19 (e.g. in-between SNP_17 and SNP_18, in between        SNP_17 and SNP17.25; or in between SNP17.25 and SNP_19, or in        between SNP17.25 and SNP_18, or in between SNP_18 and SNP_19);    -   f. any wild lettuce genome specific marker especially L.        virosa-genome specific marker, located within a distance of 12        Mb, 10 Mb, preferably within 5 Mb, of any marker selected from        SNP_17, SNP_17.25, SNP_18 and SNP_19.

Optionally, in one aspect, the introgression fragment comprises (and isdetectable by) a L. virosa accession specific marker selected from theCC or AC genotype for the Single Nucleotide Polymorphism marker VSP2 inSEQ ID NO: 28 (or in a sequence comprising substantial sequence identityto SEQ ID NO: 28) and the GG or GA genotype for the Single NucleotidePolymorphism marker VSP4 in SEQ ID NO: 29 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 29). Using the SNP markersVSP2 and VSP4 the introgression fragments comprising QTL7.1 from twodifferent types L. virosa accessions can be distinguished.

When referring to the introgression fragment being “detectable by amolecular marker assay which detects” one or more markers, this meansthat the introgression fragment comprises the resistance genotype ofthat marker.

In a further aspect, seeds, plants and plant parts or cultivated lettucecomprising an introgression fragment from a wild lettuce, such as fromL. virosa, comprising QTL6.1 and/or QTL7.1 (and optionally QTL7.2) isprovided, whereby the introgression fragment confers resistance againstNasonovia ribisnigri biotype Nr:1. In one aspect the introgressionfragment is from L. virosa, especially from L. virosa accessionscomprising Nr:1 resistance in both free choice and non-choice tests asdescribed herein. In one aspect the introgression fragment is fromaccession NCIMB42086 or is obtainable from accession NCIMB42086, orprogeny or descendants thereof.

In another aspect the introgression fragment is from a L. virosaaccession which comprises the following L. virosa accession specific SNPmarkers: the GG genotype (homozygous) or GT genotype (heterozygous) atnucleotide 71 of SEQ ID NO: 26 (or in a sequence comprising substantialsequence identity to SEQ ID NO: 26), named the VSP1 marker; and the CCgenotype (homozygous) or AC genotype (heterozygous) at nucleotide 71 ofSEQ ID NO: 28 (or in a sequence comprising substantial sequence identityto SEQ ID NO: 28), named VSP2. VSP1 and VSP2 are found in Nr:1 resistantaccessions, such as NCIMB42086.

In another aspect the introgression fragment is from a L. virosaaccession which comprises the following L. virosa accession specific SNPmarkers: the AA genotype (homozygous) or AC genotype (heterozygous) atnucleotide 71 of SEQ ID NO: 27 (or in a sequence comprising substantialsequence identity to SEQ ID NO: 27), named the VSP3 marker; and the GGgenotype (homozygous) or AG genotype (heterozygous) at nucleotide 71 ofSEQ ID NO: 29 (or in a sequence comprising substantial sequence identityto SEQ ID NO: 29), named VSP4. VSP3 and VSP4 are found in other Nr: 1resistant accessions.

Also methods for making and/or identifying and/or selecting cultivatedlettuce plants comprising an introgression from wild lettuce, such asfrom L. virosa, on chromosome 6 (comprising QTL6.1) and/or chromosome 7(comprising QTL 7.1 and/or QTL7.2) are provided, as are methods fortransferring QTLs to different cultivated lettuce plant lines orvarieties, especially to Nr:1 susceptible lettuce lines or varieties.

Definitions

The indefinite article “a” or “an” does not exclude the possibility thatmore than one of the element is present, unless the context clearlyrequires that there be one and only one of the elements. The indefinitearticle “a” or “an” thus usually means “at least one”.

“Plant variety” is a group of plants within the same botanical taxon ofthe lowest grade known, which (irrespective of whether the conditionsfor the recognition of plant breeder's rights are fulfilled or not) canbe defined on the basis of the expression of characteristics that resultfrom a certain genotype or a combination of genotypes, can bedistinguished from any other group of plants by the expression of atleast one of those characteristics, and can be regarded as an entity,because it can be multiplied without any change. Therefore, the term“plant variety” cannot be used to denote a group of plants, even if theyare of the same kind, if they are all characterized by the presence ofone or two or three loci or genes (or phenotypic characteristics due tothese specific loci or genes), but which can otherwise differ from oneanother enormously as regards the other loci or genes in the genome.

“Lettuce” or “cultivated lettuce” or “cultivated Lactuca sativa” refersherein to plants of the species Lactuca sativa L. (or seeds from whichthe plants can be grown), and parts of such plants, bred by humans forfood and having good agronomic characteristics. This includes anycultivated lettuce, such as breeding lines (e.g. backcross lines, inbredlines), cultivars and varieties of any type. Generally heading andnon-heading types of lettuce are distinguished. Heading types includefor example crisphead, butterhead and romaine (cos) types, whilenon-heading types include leaf-types. Cultivated lettuce plants are not“wild lettuce” plants or “wild Lactuca” plants, i.e. plants whichgenerally have much poorer yields and poorer agronomic characteristicsthan cultivated plants and e.g. grow naturally in wild populations.

“Wild lettuce” or “wild Lactuca” accessions refers to plants of speciesother than cultivated Lactuca sativa, such as Lactuca virosa, Lactucaserriola, Lactuca saligna, Lactuca perennis, and others. Preferably,such wild lettuce comprises or consists of Lactuca species which arecross fertile with L. sativa, optionally with the aid of embryo rescuetechniques (see Maisonneuve 1987, Agronomique 7: 313-319 and Maisonneuveet al. 1995, Euphytica 85:281-285) and/or chromosome doubling techniques(Thompson and Ryder 1961, US Dept Agric Tech Bul. 1224), or methodswhereby genes can be transferred into L. sativa via a bridge species,such as L. serriola (Eenink et al. 1982, supra).

As used herein, the term “plant” includes the seed (from which the plantcan be grown), the whole plant or any parts such as plant organs (e.g.,harvested or non-harvested leaves, etc.), plant cells, plantprotoplasts, plant cell- or tissue cultures, plant callus, plant cellclumps, plant transplants, seedlings, plant cells that are intact inplants, plant clones or micro-propagations, or parts of plants (e.g.,harvested tissues or organs), such as plant cuttings, vegetativepropagations, embryos, pollen, ovules, flowers, leaves, heads, seeds(produced on the plant after self-fertilization or cross-fertilization),clonally propagated plants, roots, stems, stalks, root tips, grafts,parts of any of these and the like, or derivatives thereof, preferablyhaving the same genetic make-up (or very similar genetic make-up) as theplant from which it is obtained. Also any developmental stage isincluded, such as seedlings, cuttings prior or after rooting, matureand/or immature plants or mature and/or immature leaves. When “seeds ofa plant” are referred to, these either refer to seeds from which theplant can be grown or to seeds produced on the plant, afterself-fertilization or cross-fertilization.

“Somatice cells” and “reproductive cells” can be distinguished, wherebysomatic cells are cells other than gametes (e.g. ovules and pollen),germ cells and gametocytes. Gametes, germ cells and gametocytes are“reproductive cells”.

“Tissue Culture” or “cell culture” refers to an in vitro compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into plant tissue. Tissue culturesand cell cultures of lettuce, and regeneration of lettuce plantstherefrom, is well known and widely published (see, e.g., Teng et al.,HortScience. 1992, 27(9): 1030-1032 Teng et al., HortScience. 1993,28(6): 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992,46(3): 287-290).

“Harvested plant material” refers herein to plant parts (e.g., leaves,leaf parts or heads detached from the whole plant) which have beencollected for further storage and/or further use.

“Harvested seeds” refers to seeds harvested from a line or variety,e.g., produced after self-fertilization or cross-fertilization andcollected.

“Harvested leaves” or “harvested heads” as used herein refers to lettuceleaves, or leaf parts or heads, i.e., the plant without the root system,for example substantially all (harvested) leaves. Leaves may be whole orcut into parts.

“Progeny” or “progenies” or “descendants” as used herein refers tooffspring, or the first and all further descendants derived from(obtained from) (derivable from or obtainable from) a plant of theinvention that comprises (retains) the one or more Nr:1 resistanceconferring QTLs in homozygous or heterozygous form and/or the Nr:1resistance phenotype described herein. Progeny may be derived byregeneration of cell culture or tissue culture, or parts of a plant, orselfing of a plant, or by producing seeds of a plant. In furtherembodiments, progeny may also encompass lettuce plants derived fromcrossing of at least one lettuce plant with another lettuce plant of thesame or another variety or (breeding) line, or wild Lactuca plants,backcrossing, inserting of a locus into a plant or mutation. A progenyis, e.g., a first generation progeny, i.e. the progeny is directlyderived from, obtained from, obtainable from or derivable from theparent plant by, e.g., traditional breeding methods (selfing and/orcrossing) or regeneration or transformation. However, the term “progeny”generally encompasses further generations such as second, third, fourth,fifth, sixth, seventh or more generations, i.e., generations of plantswhich are derived from, obtained from, obtainable from or derivable fromthe former generation by, e.g., traditional breeding methods,regeneration or genetic transformation techniques. For example, a secondgeneration progeny can be produced from a first generation progeny byany of the methods mentioned above. Also double haploid plants areprogeny.

A “plant line” or “breeding line” refers to a plant and its progenybeing highly uniform in plant phenotype. As used herein, the term“inbred line” refers to a plant line which has been repeatedly selfedand is nearly homozygous for all alleles. Thus, an “inbred line” or“parent line” refers to a plant which has undergone several generations(e.g. at least 4, 5, 6, 7 or more) of inbreeding, resulting in a plantline with a high uniformity.

“F1, F2, F3, etc.” refers to the consecutive related generationsfollowing a cross between two parent plants or parent lines. The plantsgrown from the seeds produced by crossing two plants or lines is calledthe F1 generation. Selfing the F1 plants results in the F2 generation,etc.

“Hybrid” refers to the seeds harvested from crossing one plant line orvariety with another plant line or variety, and the plants or plantparts grown from said seeds.

“F1 hybrid” plant (or F1 hybrid seed) is the generation obtained fromcrossing two non-isogenic inbred parent lines. Thus, F1 hybrid seeds areseeds from which F1 hybrid plants grow.

An “interspecific hybrid” refers to a hybrid produced from crossing aplant of one species, e.g. L. sativa, with a plant of another species,e.g. L. virosa.

“Crossing” refers to the mating of two parent plants. Equally“Cross-pollination” refers to fertilization by the union of two gametesfrom different plants.

“Selfing” refers to the self-pollination of a plant, i.e. to the unionof gametes from the same plant.

“Backcrossing” refers to a breeding method by which a trait, such as oneor more Nr: 1 resistance-conferring QTLs, can be transferred from aninferior genetic background (e.g. a wild lettuce; also referred to as“donor”) into a superior genetic background (also referred to as“recurrent parent”), e.g. cultivated lettuce. An offspring of a cross(e.g. an F1 plant obtained by crossing a wild, Nr: 1-resistant lettucewith a cultivated, Nr:1-susceptible lettuce; or an F2 plant or F3 plant,etc., obtained from selfing the F1) is “backcrossed” to the parent withthe superior genetic background, e.g. to the cultivated,Nr:1-susceptible, parent After repeated backcrossing, the trait of theinferior genetic background will have been incorporated into thesuperior genetic background. The terms “gene converted” or “conversionplant” or “single/double/triple locus conversion” in this context referto plants which are developed by backcrossing wherein essentially all ofthe desired morphological and/or physiological characteristics of therecurrent parent are recovered in addition to the one or more QTLs (e.g.the Nr:1 resistance conferring QTL6.1, QTL7.1 and/or QTL7.2) transferredfrom the donor parent.

The term “traditional breeding techniques” encompasses herein crossing,backcrossing, selfing, selection, chromosome doubling, double haploidproduction, embryo rescue, the use of bridge species, protoplast fusion,marker assisted selection, mutation breeding etc. as known to thebreeder (i.e. methods other than geneticmodification/transformation/transgenic methods), by which, for example,one or more Nr:1-resistance conferring QTLs, referred herein to asQTL6.1, QTL7.1 and/or QTL7.2, can be obtained, identified, selected,and/or transferred.

“Regeneration” refers to the development of a plant from in vitro cellculture or tissue culture or vegetative propagation.

“Vegetative propagation”, “vegetative reproduction” or “clonalpropagation” are used interchangeably herein and mean the method oftaking part of a plant and allowing that plant part to form at leastroots where plant part is, e.g., defined as or derived from (e.g. bycutting off) leaf, pollen, embryo, cotyledon, hypocotyl, cells,protoplasts, meristematic cell, root, root tip, pistil, anther, flower,shoot tip, shoot, stem, fruit, and petiole. When a whole plant isregenerated by vegetative propagation, it is also referred to as a“vegetative propagation” or a “vegetatively propagated plant”.

“Single (or double or triple) locus converted (conversion) plant” refersto plants which are developed by plant breeding techniques comprising orconsisting of backcrossing, wherein essentially all of the desiredmorphological and/or physiological characteristics of a lettuce plantare recovered in addition to the characteristics of the single locus (ortwo or three loci) having been transferred into the plant via e.g. thebackcrossing technique.

“Transgene” or “chimeric gene” refers to a genetic locus comprising aDNA sequence which has been introduced into the genome of a lettuceplant by transformation. A plant comprising a transgene stablyintegrated into its genome is referred to as “transgenic plant”.

“Substantially equivalent” or “not significantly different” or “notstatistically significantly different” refers to a characteristic that,when compared e.g. between two plant lines or varieties, is equivalentor almost identical. In other words, a characteristic being“substantially equivalent” between two plant lines or varieties meansthat the mean value for said characteristic differs less than 10% (e.g.9, 8, 7, 6, 5, 4, 3, 2, 1% or less) and the statistical significance ofthis difference is not having a p≥0.05 using ANOVA.

“Average” refers herein to the arithmetic mean. The term “mean” refersto the arithmetic mean of several measurements. The skilled personunderstands that the phenotype of a plant line or variety depends tosome extent on growing conditions and that, therefore, arithmetic meansof at least 10, 15, 20, 30, 40, 50 or more plants are measured,preferably in randomized experimental designs with several replicatesand suitable control plants grown under the same conditions in the sameexperiment.

“Statistically significant” or “statistically significantly” differentor “significantly” different refers to a characteristic of a plant lineor variety, such as Nr: 1 resistance, that, when compared to a suitablecontrol (e.g. herein the genetic control line lacking the QTLs, or a Nr:1 susceptible control variety, such as Mafalda) show a statisticallysignificant difference in that characteristic (e.g. the p-value is lessthan 0.05, p<0.05, using ANOVA) from the (mean of the) control. So, e.g.a plant line or variety or genotype which has on average “statisticallysignificantly fewer” aphids or “significantly fewer” aphids than thecontrol, is a plant wherein the difference in average aphid number isstatistically significant from the control plant.

“Lettuce aphid” refers to aphids of the species Nasonovia ribisnigri.

“Biotype Nr:0” refers to the lettuce aphid biotype against which the Nrgene from IVT280 provides resistance, i.e. lettuce aphids of thisbiotype are unable to feed and reproduce on varieties comprising the Nrgene, such as Mafalda (Nunhems), Barcelona (Nunhems), or others.

“Biotype Nr:1” refers to the lettuce aphid biotype against which the Nrgene from IVT280 does not provide resistance. Thus, lettuce aphids ofthis biotype can feed and reproduce on varieties and lines comprisingthe Nr gene, such as Mafalda (Nunhems), Barcelona (Nunhems), or others.

When referring to resistance tests carried out under controlledenvironmental conditions (e.g. in climate cells), preferably clonalcolonies of single, selected aphids of biotype Nr:0 or Nr:1 are referredto. These are also referred to as aphid “isolates” herein.

A wild or cultivated lettuce plant line, variety or accessions is saidto be a “Nr:0 resistant plant”, or a “plant resistant against biotypeNr:0”, or a plant having “Nr:0 resistance”, or an “Nr:0 resistancephenotype”, if reproduction (average number of aphids per plant, countedperiodically, e.g. after about 7, 14, 21, 28, 35 and/or more days, afterinfestation with aphids of biotype Nr:0) of biotype Nr:0 isstatistically significantly reduced compared to control plants lackinglettuce aphid resistance genes, such as susceptible varieties Salinas(synonym Saladin; originally developed by breeder Ryder E.J., USDA, ARS,California, USA), or others. A significant reduction in reproduction ofbiotype Nr:0 can be determined using e.g. a greenhouse test or cagedfield test, as known in the art, e.g. as described in McCreight and Liu,2012, HortScience 47(2), in Materials and Methods, or others. Thegreenhouse test or caged field test may be a free-choice (aphids areable to feed and reproduce on several plant lines or varieties) and/or anon-choice test (aphids are only able to feed and reproduce on one plantline or variety). Alternatively, open field tests may be carried out,whereby the natural infestation of susceptible controls, such as varietySalinas (synonym Saladin), is monitored and when infestation on thecontrol is numerous, aphids are counted on test plants and controls. Theterm encompasses both “partial Nr:0 resistance” (as in PI491093described in McCreight and Liu, 2012) and “complete Nr:0 resistance” (asin IVT280, see idem McCreight and Liu 2012). On completely Nr:0resistant plants virtually no aphids of biotype Nr:0 feed and reproduceat the weekly time points measured post-inoculation or post-infestation.

A wild or cultivated lettuce plant line, variety or accessions is saidto be a “Nr:1 resistant plant”, or a “plant resistant against biotypeNr:1”, or a plant having “Nr:1 resistance”, or an “Nr:1 resistancephenotype”, or a “plant having significantly reduced susceptibility”, or“significantly enhanced resistance”, if the average number of aphids ofbiotype Nr:1 per plant, counted at one or more time-points afterinfestation with aphids of biotype Nr:1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or more weeks after transplant of seedlings into thefield; and/or after plants have reached the 3-4 true leaf stage andthereafter, i.e. when plants have reached 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% of the final adult size) is statistically significantlyreduced compared to control plants lacking lettuce aphid resistancegenes (such as variety Salinas (synonym Saladin)) and/or compared tocontrol plants having the Nr resistance gene, such as variety Mafalda(Nunhems) and/or the genetic control lacking the introgressionfragment(s) but otherwise being genetically identical or geneticallyvery similar to the plant comprising the introgression fragment(s). Inone aspect, a significant reduction refers to the average number ofaphids on the Nr:1 resistant plant being at most 50%, 49%, 48%, 47%,45%, preferably at most 40%, preferably at most 30%, 20% or 10%, morepreferably at most 5%, 3%, 2%/0, or 1% of the average number of aphidsfound on a Nr: 1 susceptible variety, such as variety Mafalda (or otherNr: 1 susceptible varieties comprising the Nr gene), or on the geneticcontrol, when grown under the same conditions. In one embodiment, plantsare free or virtually free (less than an average of 10, 9, 8, 7, 6, 5,4, 3 aphids per plant line or variety) of Nr:1 aphids. Nr:1 resistancecan be determined in Nr:1 resistance assays as defined and as describedin the Examples.

A “Nr: 1-resistance assay” may be either a non-choice test (aphids areonly able to feed and reproduce on one plant line or variety) and/or a(free-) choice test, as e.g. described in the Examples. The choice ornon-choice tests may be in controlled environments, such as climatecells, or in the field (open field for choice or caged field fornon-choice tests). Choice-tests for resistance refers to tests where theaphids can choose among different plant genotypes for feeding andreproduction. Generally, choice-tests are used to identify antixenosis(non-preference) resistance, i.e. resistance caused by factors that makea plant genotype less attractive. Non-choice tests for resistance refersto tests where the aphids cannot choose among different plant genotypesfor feeding and reproduction, but are only allowed to feed and reproduceon one genotype. This allows antibiosis to be detected. On Nr: 1susceptible control plants, such as Mafalda, the aphid is able toreproduce to more than about 50, 100, 150, 200, 250, 300 or more aphids.Resistance effective under non-choice conditions affects the insectsthemselves, e.g. they die, produce fewer offspring or grow more slowly.

A genetic element, a locus, an introgression fragment or a gene orallele conferring a trait (such as one or more QTLs conferringresistance against N. ribisnigri biotype Nr: 1) is said to be“obtainable from” or can be “obtained from” or “derivable from” or canbe “derived from” or “as present in” or “as found in” a plant or seed ifit can be transferred from the plant or seed in which it is present intoanother plant or seed in which it is not present (such as a line orvariety) using traditional breeding techniques without resulting in aphenotypic change of the recipient plant apart from the addition of thetrait (Nr:1 resistance) conferred by the genetic element, locus,introgression fragment, gene or allele. The terms are usedinterchangeably and the genetic element, locus, introgression fragment,gene or allele can thus be transferred into any other genetic backgroundlacking the trait. Not only seeds deposited and comprising the geneticelement, locus, introgression fragment, gene or allele can be used, butalso progeny/descendants from such seeds which have been selected toretain the genetic element, locus, introgression fragment, gene orallele, can be used and are encompassed herein, such as commercialvarieties developed from the deposited seeds or from descendantsthereof. Whether a plant (or genomic DNA, cell or tissue of a plant)comprises the same genetic element, locus, introgression fragment, geneor allele as obtainable from the deposited seeds can be determined bythe skilled person using one or more techniques known in the art, suchas phenotypic assays, whole genome sequencing, molecular marker analysis(e.g. using one or more or all of the marker disclosed herein), traitmapping, chromosome painting, allelism tests and the like, orcombinations of techniques.

The term “allele(s)” means any of one or more alternative forms of agene at a particular locus, all of which alleles relate to one trait orcharacteristic at a specific locus. In a diploid cell of an organism,alleles of a given gene are located at a specific location, or locus(loci plural) on a chromosome. One allele is present on each chromosomeof the pair of homologous chromosomes. A diploid plant species maycomprise a large number of different alleles at a particular locus.These may be identical alleles of the gene (homozygous) or two differentalleles (heterozygous).

The term “gene” means a (genomic) DNA sequence comprising a region(transcribed region), which is transcribed into a messenger RNA molecule(mRNA) in a cell, and an operably linked regulatory region (e.g. apromoter). Different alleles of a gene are thus different alternativeforms of the gene, which may be in the form of e.g. differences in oneor more nucleotides of the genomic DNA sequence (e.g. in the promotersequence, the exon sequences, intron sequences, etc.), mRNA and/or aminoacid sequence of the encoded protein.

“Allelism test” refers to a genetic test whereby it can be testedwhether two phenotypes, e.g. two Nr:1 resistances, seen in two plantlines or varieties are determined by the same gene or by differentgenes. For example, the plants to be tested are crossed with each other,the F1 is selfed and the segregation of the phenotypes amongst the F2progeny is determined. The ratio of segregation indicates if the genesare allelic.

The term “locus” (loci plural) means a specific place or places or asite on a chromosome where for example a gene or genetic marker isfound. The Nr: 1 resistance locus (or Nr1 resistance-conferringlocus/loci) is/are, thus, the location in the genome of wild lettuce,especially Lactuca virosa accession, such as (but not limited to) NCIMB42086, where the Nr: 1-resistance conferring QTL(s) is/are found onchromosome 6 (QTL6.1) and/or on chromosome 7 (QTL7.1 and/or QTL7.2). Incultivated lettuce according to the invention one or more QTLsconferring Nr: 1 resistance are introgressed from wild lettuceaccessions which comprise one or more of the QTLs, such as the wild L.virosa accession deposited under accession numbers NCIMB 42086.

A “quantitative trait locus”, or “QTL” is a chromosomal locus thatencodes for one or more alleles that affect the expressivity of acontinuously distributed (quantitative) phenotype. The resistanceconferring quantitative trait loci are named herein QTL6.1, QTL7.1 andQTL7.2.

“Lettuce genome” and “physical position on the lettuce genome” and“chromosome 6” and/or on “chromosome 7” refers to the physical genome ofcultivated lettuce, world wide web at lgr.genomecenter.ucdavis.edu(Lettuce version 3.2 Database, comprising chromosome 6, designatedLsat_1_v4_1g6, and chromosome 7, designated Lsat_1_v4_1g7), and thephysical chromosomes and the physical position on the chromosomes. So,for example SNP_01 is located at the nucleotide (or ‘base’) positionedphysically at nucleotide 60,688,939 of chromosome 6, which has aphysical size from 0 to 244.7 Mb. Likewise, SNP_08 is located at thenucleotide (or ‘base’) positioned at 72,772,104 of chromosome 7, whichchromosome has a physical size from 0 to 242.9 Mb.

“Physical distance” between loci (e.g. between molecular markers and/orbetween phenotypic markers) on the same chromosome is the actuallyphysical distance expressed in bases or base pairs (bp), kilo bases orkilo base pairs (kb) or megabases or mega base pairs (Mb).

“Genetic distance” between loci (e.g. between molecular markers and/orbetween phenotypic markers) on the same chromosome is measured byfrequency of crossing-over, or recombination frequency (RF) and isindicated in centimorgans (cM). One cM corresponds to a recombinationfrequency of 1%. If no recombinants can be found, the RF is zero and theloci are either extremely close together physically or they areidentical. The further apart two loci are, the higher the RF.

“Introgression fragment” or “introgression segment” or “introgressionregion” refers to a chromosome fragment (or chromosome part or region)which has been introduced into another plant of the same or relatedspecies by crossing or traditional breeding techniques, such asbackcrossing, i.e. the introgressed fragment is the result oftraditional breeding methods referred to by the verb “to introgress”(such as backcrossing). In lettuce, wild lettuce accessions may be usedto introgress fragments of the wild genome (e.g. L. virosa) into thegenome of cultivated lettuce, L. sativa. Such a cultivated lettuce plantthus has a “genome of cultivated L. sativa”, but comprises in the genomea fragment (or two or three fragments) of a wild lettuce, e.g. anintrogression fragment (or two or three) of a related wild Lactucagenome, such as L. virosa. It is understood that the term “introgressionfragment” never includes a whole chromosome, but only a part of achromosome. The introgression fragment can be large, e.g. even half of achromosome, but is preferably smaller, such as about 80 Mb, 74 Mb, 73Mb, 70 Mb, 50 Mb, 30 Mb, 20 Mb, 15 Mb or less, such as about 10 Mb orless, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less, about6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb orless, about 2 Mb or less, about 1 Mb (equals 1,000,000 base pairs) orless, or about 0.5 Mb (equals 500,000 base pairs) or less, such as about200,000 bp (equals 200 kilo base pairs) or less, about 100,000 bp (100kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) orless.

“Uniformity” or “uniform” relates to the genetic and phenotypiccharacteristics of a plant line or variety. Inbred lines are geneticallyhighly uniform as they are produced by several generations ofinbreeding.

The term “Nr:1-allele” or “Nr:1 resistance allele”, refers to an allelefound at the locus QTL6.1 or QTL7.1 or QTL7.2 which in one aspect of theinvention is introgressed into cultivated lettuce (onto cultivated L.sativa chromosome 6 and/or 7) from a wild lettuce, especially from a L.virosa accession. The term “Nr:1-allele”, thus, also encompassesNr:1-alleles obtainable from different wild lettuce accessions. When oneor two Nr:1-alleles are present at a specific locus in the genome (i.e.in heterozygous or homozygous form, respectively), the plant line orvariety has significantly enhanced Nr:1 resistance compared to thegenetic control lacking the QTL. In cultivated lettuce plant lacking theintrogression fragment, the L. sativa allele found at the same locus onchromosome 6 and/or 7 is herein referred to as “wild type” allele (wt).Thus, a cultivated lettuce susceptible to Nr:1 and lacking the QTLs onchromosome 6 and 7 is designated wt/wt, whereas QTL6.1/wt and/orQTL7.1/wt and/or QTL7.2/wt plants, and QTL6.1/QTL6.1 and/orQTL7.1/QTL7.1 and/or QTL7.2/QTL7.2 plants, are cultivated lettuce plantswhich possess the QTLs in heterozygous or homozygous form, respectively.The genotype of the SNP markers provided herein is also indicative ofthe wild type genotype or of the introgression fragment comprising theQTLs being in homozygous or heterozygous form. E.g. the genotype ofSNP_01 indicative of QTL6.1 is ‘AT’ (indicative of QTL6.1/wt) or ‘AA’(indicative of QTL6.1/QTL6.1) while the genotype indicative of the wildtype is ‘T’ (wt/wt). See elsewhere herein for all other SNPs. Thus, whenreference is made to a SNP marker herein or a SNP genotype, the genotypeof the marker indicative of the introgression fragment comprising theQTL conferring Nr:1 resistance is referred to (in homozygous orheterozygous form).

“Variant” or “orthologous” sequences or “variant QTL6.1, QTL7.1 orQTL7.2” refers to QTLs (QTL6.1, QTL7.1 or QTL7.2), or introgressionfragment(s) comprising these, which are derived from different wildlettuce plants (especially different L. virosa plants or accessions)than the QTL6.1, QTL7.1 and QTL7.2 (and genomic region comprising these)present in NCIMB42086, but which variants comprise one or more or all ofthe SNPs linked to QTL6.1, QTL7.1 and/or QTL7.2 and wherein the variantgenomic sequence comprises substantial sequence identity to the SEQ IDNO: comprising the SNP (any one of SEQ ID NO: 1-22, SNP1.23, SNP2.24,SNP17.25, VSP1 to VSP4), i.e. at least 85%, 90%, 95%, 98%, 99% sequenceidentity or more. Thus, when reference herein is made to a certain SNPgenotype in a specific genomic sequence (selected from SEQ ID NO: 1 toSEQ ID NO: 22, SNP1.23, SNP2.24, SNP17.25, VSP1 to VSP4), thisencompasses also the SNP genotype in variants of the genomic sequence,i.e. the SNP genotype in a genomic sequence comprising at least 85%,90%/0, 95%, 98%, 99% sequence identity or more to the sequence referredto (selected from SEQ ID NO: 1 to SEQ ID NO: 22, SNP1.23, SNP2.24,SNP17.25, VSP1 to VSP4). Thus any reference herein to any one of SEQ IDNO: 1 to 22, SNP1.23, SNP2.24, SNP17.25, VSP1 to VSP4, in one aspectalso encompasses a variant of any one of SEQ ID NO: 1 to 22, SNP1.23,SNP2.24, SNP17.25, VSP1 to VSP4, said variant comprising at least 85%,90%/0, 95%, 98%, 99% sequence identity or more to said sequence.

“Genetic control” is a lettuce line, variety or hybrid which has thesame or very similar cultivated genome as the cultivated lettuce plantcomprising the introgression on chromosome 6 (QTL6.1) and/or 7 (QTL7.1and/or QTL7.2), except that it lacks said introgressions on chromosome 6and 7, i.e. chromosomes 6 and 7 are “wild type” (wt/wt), i.e. cultivatedlettuce genome.

The term “marker assay” refers to a molecular marker assay which can beused to test whether on cultivated L. sativa chromosome 6 and/or 7 anintrogression from a wild lettuce is present which introgressionfragment comprises the Nr:1 resistance conferring QTL (QTL6.1 and/orQTL7.1 and/or QTL7.2, or a variant of these) (or whether a wild lettucecomprises the QTL6.1 and/or QTL7.1 and/or QTL7.2 or variants of these intheir genome), by determining the genotype of any one or more markerslinked to the QTL6.1 (or a variant), e.g. the genotype of one or moreSNP markers selected from SNP_01 to SNP_07, and/or any wild lettuce(especially L. virosa) genome-specific marker in-between SNP markersSNP_01 and SNP_07, and/or within 7 cM or within 5 cM of any one of thesemarkers, and/or within 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.1 Mb, 50 kb, 20kb or less of any one of these markers; alternatively by determining thegenotype of any one or more markers linked to the QTL6.1 (or a variant),e.g. the genotype of one or more SNP markers selected from SNP1.23,SNP_02, SNP2.24 and SNP_03 (optionally also VSP1 or VSP3), and/or anywild lettuce (especially L. virosa) genome-specific marker in-betweenSNP markers SNP1.23, SNP_02, SNP2.24 and SNP_03 (optionally also VSP1 orVSP3), and/or within 7 cM or within 5 cM of any one of these markers,and/or within 12 Mb, 10 Mb, 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.1 Mb, 50kb, 20 kb or less of any one of these markers; and/or the genotype ofany one or more markers linked to the QTL7.1 (or a variant), e.g. thegenotype of one or more SNP markers selected from SNP_15 to SNP_22,and/or any wild lettuce genome-specific marker in-between SNP markersSNP_15 and SNP_22, and/or within 7 cM or within 5 cM of any one of thesemarkers, and/or within 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.1 Mb, 50 kb, 20kb or less of any one of these markers; alternatively by determining thegenotype of any one or more markers linked to the QTL7.1 (or a variant),e.g. the genotype of one or more SNP markers selected from SNP_17,SNP_17.25, SNP_18 and SNP_19 (optionally also VSP2 or VSP4), and/or anywild lettuce (especially L. virosa) genome-specific marker in-betweenSNP markers SNP_17, SNP_17.25, SNP_18 and SNP_19 (optionally also VSP2or VSP4), and/or within 7 cM or within 5 cM of any one of these markers,and/or within 12 Mb, 10 Mb, 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.1 Mb, 50kb, 20 kb or less of any one of these markers; and/or the genotype ofany one or more markers linked to the QTL7.2 (or a variant), e.g. thegenotype of one or more SNP markers selected from SNP_08 to SNP_14,and/or any wild lettuce genome-specific marker in-between SNP markersSNP_08 and SNP_14, and/or within 7 cM or within 5 cM of any one of thesemarkers, and/or within 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.1 Mb, 50 kb, 20kb or less of any one of these markers A marker “in between” two markersis physically located in between the markers on the chromosome.

A “recombinant chromosome” refers to a chromosome having a new geneticmakeup arising through crossing-over between homologous chromosomes,e.g. a “recombinant chromosome 6” or a “recombinant chromosome 7”, i.e.a chromosome 6 or 7 which is not present in either of the parent plantsand arose through a rare crossing-over event between homologouschromosomes of a chromosome 6 or 7 pair, respectively. Herein, forexample, recombinant lettuce chromosomes 6 and 7 are provided, eachcomprising a Nr:1 resistance QTL.

“Marker assisted selection” or “MAS” is a process of using the presenceof molecular markers, which are genetically linked to a particular locusor to a particular chromosome region (e.g. introgression fragment), toselect plants for the presence of the specific locus or region(introgression fragment). For example, a molecular marker geneticallylinked to a Nr:1 QTL, can be used to detect and/or select lettuce plantscomprising the Nr: 1 QTL on chromosome 6 and/or 7. The closer thegenetic linkage of the molecular marker to the locus (e.g. about 7 cM, 6cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM or less), the less likely it isthat the marker is dissociated from the locus through meioticrecombination. Likewise, the closer two markers are linked to each other(e.g. within 7 cM or 5 cM, 4 cM, 3 cM, 2 cM, 1 cM or less) the lesslikely it is that the two markers will be separated from one another(and the more likely they will co-segregate as a unit).

A marker “within 7 cM or within 5 cM” of another marker refers to amarker which genetically maps to within the 7 cM or 5 cM region flankingthe marker (i.e. either side of the marker). Similarly, a marker within10 Mb, 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb,0.1 Mb, 50 kb, 20 kb, 10 kb, 5 kb or less of another marker refers to amarker which is physically located within the 5 Mb, 3 Mb, 2.5 Mb, 2 Mb,1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, 50 kb, 20 kb, 10 kb, 5 kbor less, of the genomic DNA region flanking the marker (i.e. either sideof the marker).

“LOD-score” (logarithm (base 10) of odds) refers to a statistical testoften used for linkage analysis in animal and plant populations. The LODscore compares the likelihood of obtaining the test data if the two loci(molecular markers loci and/or a phenotypic trait locus) are indeedlinked, to the likelihood of observing the same data purely by chance.Positive LOD scores favor the presence of linkage and a LOD scoregreater than 3.0 is considered evidence for linkage. A LOD score of +3indicates 1000 to 1 odds that the linkage being observed did not occurby chance.

An “isolated nucleic acid sequence” or “isolated DNA” refers to anucleic acid sequence which is no longer in the natural environment fromwhich it was isolated, e.g. the nucleic acid sequence in a bacterialhost cell or in the plant nuclear or plastid genome.

A “host cell” or a “recombinant host cell” or “transformed cell” areterms referring to a new individual cell (or organism) arising as aresult of at least one nucleic acid molecule, having been introducedinto said cell. The host cell is preferably a plant cell or a bacterialcell. The host cell may contain the nucleic acid as anextra-chromosomally (episomal) replicating molecule, or comprises thenucleic acid integrated in the nuclear or plastid genome of the hostcell, or as introduced chromosome, e.g. minichromosome.

“Sequence identity” and “sequence similarity” can be determined byalignment of two peptide or two nucleotide sequences using global orlocal alignment algorithms. Sequences may then be referred to as“substantially identical” or “essentially similar” when they areoptimally aligned by for example the programs GAP or BESTFIT or theEmboss program “Needle” (using default parameters, see below) share atleast a certain minimal percentage of sequence identity (as definedfurther below). These programs use the Needleman and Wunsch globalalignment algorithm to align two sequences over their entire length,maximizing the number of matches and minimises the number of gaps.Generally, the default parameters are used, with a gap creationpenalty=10 and gap extension penalty=0.5 (both for nucleotide andprotein alignments). For nucleotides the default scoring matrix used isDNAFULL and for proteins the default scoring matrix is Blosum62(Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignmentsand scores for percentage sequence identity may for example bedetermined using computer programs, such as EMBOSS as available on theworld wide web under ebi.ac.uk/Tools/psalemboss_needle/). Alternativelysequence similarity or identity may be determined by searching againstdatabases such as FASTA, BLAST, etc., but hits should be retrieved andaligned pairwise to compare sequence identity. Two proteins or twoprotein domains, or two nucleic acid sequences have “substantialsequence identity” if the percentage sequence identity is at least 85%,90%, 95%, 98%, 99% or more (e.g. at least 99.1, 99.2 99.3 99.4, 99.5,99.6, 99.7, 99.8, 99.9 or more (as determined by Emboss “needle” usingdefault parameters, i.e. gap creation penalty=10, gap extensionpenalty=0.5, using scoring matrix DNAFULL for nucleic acids an Blosum62for proteins).

When reference is made to a nucleic acid sequence (e.g. DNA or genomicDNA) having “substantial sequence identity to” a reference sequence orhaving a sequence identity of at least 80%, e.g. at least 85%, 90%/0,95%, 98%, 99%, 99.2%, 99.5%, 99.9% nucleic acid sequence identity to areference sequence, in one embodiment said nucleotide sequence isconsidered substantially identical to the given nucleotide sequence andcan be identified using stringent hybridisation conditions. In anotherembodiment, the nucleic acid sequence comprises one or more mutationscompared to the given nucleotide sequence but still can be identifiedusing stringent hybridisation conditions.

“Stringent hybridisation conditions” can be used to identify nucleotidesequences, which are substantially identical to a given nucleotidesequence. Stringent conditions are sequence dependent and will bedifferent in different circumstances. Generally, stringent conditionsare selected to be about 5° C. lower than the thermal melting point (Tm)for the specific sequences at a defined ionic strength and pH. The Tm isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridises to a perfectly matched probe. Typicallystringent conditions will be chosen in which the salt concentration isabout 0.02 molar at pH 7 and the temperature is at least 60° C. Loweringthe salt concentration and/or increasing the temperature increasesstringency. Stringent conditions for RNA-DNA hybridisations (Northernblots using a probe of e.g. 100 nt) are for example those which includeat least one wash in 0.2×SSC at 63° C. for 20 min, or equivalentconditions. Stringent conditions for DNA-DNA hybridisation (Southernblots using a probe of e.g. 100 nt) are for example those which includeat least one wash (usually 2) in 0.2×SSC at a temperature of at least50° C., usually about 55° C., for 20 min, or equivalent conditions. Seealso Sambrook et al. (1989) and Sambrook and Russell (2001).

FIGURES

FIG. 1: A. Graph showing the average number of aphids (German isolate ofNr: 1) on susceptible control variety Mafalda and on NCIMB 42086 in anon-choice test, at 7, 14, 21, 28 and 35 days post inoculation. B. showsthe same graph as A., but on a more detailed scale.

FIG. 2: A. Graph showing the average number of aphids on NCIMB 42086 ina free-choice test, at 7, 14, 21, 28 and 35 days post inoculation. A.relates to a German Nr:1 isolate from the Pfalz region, while B. relatesto a French Nr: 1 isolate from the Perpignan area.

FIG. 3A: Schematic graph (not to scale) of chromosome 6 and 7 of L.sativa comprising an introgression fragment (exemplified by the fat bar,which may be longer or shorter than drawn herein) from L. virosaaccession NCIMB42086 on chromosome 6 (comprising QTL6.1) and twointrogression fragments from L. virosa accession NCIMB42086 onchromosome 7 (comprising QTL7.1 and QTL7.2), as well as the SNP markersindicative of the introgression fragments and their physical position onthe lettuce genome. The number of stars (*) indicates the LOD-scorebeing significant (one star) or highly significant (two or three stars).

FIG. 3B: Schematic graph (not to scale) of chromosome 6 and 7 of L.sativa comprising an introgression fragment (gray bar) from a wild L.virosa (e.g. from accession NCIMB42086) on chromosome 6 (comprisingQTL6.1 or a variant thereof) and on chromosome 7 (comprising QTL7.1 or avariant thereof). The * indicates SNP markers which are specific for twowild L. virosa accessions, SNP markers VSP1 and VSP3 on chromosome 6 andVSP2 and VSP4 on chromosome 7 (whereby VSP1 and VSP2 are specific forone of the accessions and VSP3 and VSP4 are specific for the otheraccession.

FIG. 4: Results of the non-choice field assay carried out in Murcia,Spain, showing zero aphids on NCIMB42086 compared to >300 aphids on theNr:0 resistant variety Mafalda.

DETAILED DESCRIPTION

The present invention relates to a cultivated lettuce plant, Lactucasativa, comprising one or two or three QTLs introgressed from wildlettuce, wherein said QTLs confer resistance against N. ribisnigribiotype 1 (Nr:1). In particular, the Nr:1 resistance is conferred by anintrogression fragment on cultivated lettuce chromosome 6 (comprisingQTL6.1) and/or 7 (comprising QTL 7.1 and/or QTL7.2), wherein saidintrogression fragment is from a wild lettuce plant, in particular aplant of the species Lactuca virosa.

Thus, in one aspect a Lactuca sativa plant is provided comprising anintrogression fragment from a wild lettuce plant on chromosome 6 and/oron chromosome 7, wherein said introgression fragment on chromosome 6comprises a Quantitative Trait Locus (QTL) referred to as QTL6.1 andwherein said introgression fragment on chromosome 7 comprises aQuantitative Trait Locus referred to as QTL7.1 and/or QTL7.2, andwherein said introgression fragment on chromosome 6 and/or 7 confersresistance against Nasonovia ribisnigri biotype 1 (Nr: 1). Each one ofthe introgression fragments may be in homozygous or heterozygous form.In one aspect, the introgression fragment(s) is/are in homozygous form.In one aspect, where the plant comprises more than one introgressionfragment, i.e. two or three fragments, these originate from the samewild lettuce accession. In one embodiment of the invention this wildlettuce accession is a L. virosa accession (such as NCIMB 42086 orprogeny thereof). In one aspect the wild accession is a L. virosaaccession selected from an accession comprising L. virosa accessionspecific markers VSP1 and VSP2; or an accession comprising L. virosaaccession specific markers VSP3 and VSP4.

When reference is made herein to an introgression fragment on chromosome6 and/or 7 having Nr:1 resistance conferring QTL this encompassesvarious sizes of introgression fragments, e.g. the fragment comprisingall SNP markers (SNP_01 to SNP_07, or in the alternative SNP1.23,SNP_02, SNP2.24 and SNP_03, or any marker in between these, for thefragment on chromosome 6 (comprising QTL6.1); SNP_08 to SNP_14, or anymarker in between these, for the fragment on chromosome 7 (comprisingQTL7.2); SNP_15 to SNP_22, or in the alternative SNP_17, SNP17.25,SNP_18 and SNP_19, or any marker in between these, for the fragment onchromosome 7, comprising QTL7.1), but also smaller introgressionfragments (comprising e.g. 1, 2, 3 or 4 of the SNP markers), wherehowever the fragment remains large enough to confer Nr:1 resistance whenthe introgression fragment(s) is/are in heterozygous or homozygous formin the cultivated lettuce genome.

When referring to the SNP markers herein, which are indicative of thepresence of the introgression fragment (and the Nr:1 QTL present on theintrogression fragment), it is understood that the SNP genotype which isindicative of the introgression fragment is referred to, i.e. the SNPgenotype as provided in Table 1, 2 and 3, or in Table 4, 5, 6 and 7, andherein below. It is noted that the SNP marker genotype can distinguishbetween the introgression fragment being in homozygous or heterozygousform, as shown in these Tables. In homozygous form the nucleotide isidentical, while in heterozygous form the nucleotide is not identical.The SNP genotype of the ‘wild type’ chromosome lacking the introgressionfragment is the other genotype, also listed in Tables 1-3 and Tables 4-7(under genotype of L. sativa parent). So, e.g. the genotype of SNP_01indicative of the introgression fragment comprising QTL6.1 is ‘AT’(QTL6.1/wt, i.e. heterozygous for the resistance conferring QTL) or ‘AA’(QTL6.1/QTL6.1, i.e. homozygous for the resistance conferring QTL) whilethe SNP genotype indicative of the wild type/genetic control (lackingthe introgression fragment) is ‘TT’ (wt/wt). Thus, when referring to aplant or plant part (e.g. cell) comprising the introgression fragment inhomozygous or heterozygous form, it is understood that the SNP markerslinked to the introgression fragment have the corresponding SNPgenotype. For example, a plant according to the invention which ishomozygous for the introgression fragment comprising QTL6.1 comprisesthe SNP markers in homozygous form.

So in one aspect, a cultivated L. sativa plant is provided comprising anintrogression fragment on chromosome 6 and/or on chromosome 7 inhomozygous or heterozygous form, wherein said introgression fragmentconfers resistance against biotype Nr: 1. In a preferred aspect, one,two or all three of the introgression fragments are in homozygous form(and the SNP marker(s) indicative of the QTL are homozygous for thementioned nucleotide).

The resistance against Nr:1 is phenotypically expressed as a(statistically) significantly lower average number of aphids of biotypeNr:1 on the plants of the cultivated lettuce plant line or varietycomprising the introgression fragment(s) on chromosome 6 (comprisingQTL6.1) and/or 7 (comprising QTL7.1 and/or 7.2) in homozygous orheterozygous form compared to the control line or variety lacking theintrogression fragment on chromosome 6 and 7 when grown under the sameenvironment. The control line or variety is a cultivated lettuce line orvariety which is susceptible against Nr: 1. In one aspect is it selectedfrom a variety which is susceptible against Nr:0 and Nr:1 (i.e. lackingN. ribisnigri resistance). In another preferred aspect it is a line orvariety comprising Nr:0 resistance conferred by the dominant Nr gene,such as variety Mafalda (or others, such as Susana, Sylvesta, Veronique,and many others, see the world wide web at nunhems.nl, where varietieswith Nr:0 resistance are indicated as ‘HR’). In yet another aspect it isthe genetic control line or variety.

Thus, different cultivated lettuce plants are provided herein, whicheither comprise an introgression fragment on chromosome 6 (comprisingQTL6.1 or a variant thereof) in homozygous or heterozygous form; orwhich comprise an introgression fragment on chromosome 7 (comprisingQTL7.1 or a variant thereof) in homozygous or heterozygous form; orwhich comprise an introgression fragment on chromosome 7 (comprisingQTL7.2 or a variant thereof) in homozygous or heterozygous form; orwhich comprise two introgression fragments (QTL6.1 and QTL7.1 orvariants of either of these; or QTL6.1 and QTL7.2 or variants of eitherof these; or QTL7.1 and QTL7.2 or variants of either of these; whereinthe two QTLs can independently from each other be in homozygous orheterozygous form) or all three introgression fragments (QTL6.1, QTL7.1and QTL7.2 or variants of any of these), any one of these three QTLsindependently being in homozygous or in heterozygous form.

The plants of the invention therefore comprise a genome of cultivatedlettuce, with one or two recombinant chromosomes 6 and/or with one ortwo recombinant chromosomes 7. The recombinant chromosomes comprise afragment of a wild lettuce (especially of L. virosa; in one aspect of L.virosa accession NCIMB42086 or progeny thereof, or from another L.virosa, such as an accession comprising VSP1 and VSP2 or comprising VSP3and VSP4), which is easily distinguishable from the cultivated lettucegenome by molecular marker analysis (using e.g. the markers providedherein), whole genome sequencing, chromosome painting and similartechniques.

In one aspect the presence of the introgression fragment(s) onchromosomes 6 and/or 7 in the genome of the plant or plant cell or planttissue (or in the DNA extracted therefrom) is detectable by a molecularmarker assay which detects one or more molecular markers of theintrogression fragment(s). However, as mentioned, other techniques maybe used, e.g. the SNP genotype of the markers may also be determined bysequencing or by using alternative markers located in-between the SNPmarkers provided herein or within 7 cM, or within 5 cM, of a markerprovided herein; or within 10 Mb, 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, 50 kb, 20 kb, 10 kb, 5 kb or less ofa marker provided herein.

Lettuce Plants Comprising an Introgression Fragment on Chromosome 6(Comprising QTL 6.1 or a Variant Thereof)

Based on the first QTL mapping results, the following cultivated lettuceplants are encompassed herein.

In one aspect the introgression fragment on chromosome 6 is detectableby a molecular marker assay which detects at least 1, preferably atleast 2 or 3, or at least 4, 5, 6, or 7 of the markers selected from thegroup consisting of:

-   -   a) the AA or AT genotype for the Single Nucleotide Polymorphism        marker SNP_01 in SEQ ID NO: 1 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:1);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:2);    -   c) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:3);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_04 in SEQ ID NO: 4 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:4);    -   e) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:5);    -   f) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_06 in SEQ ID NO: 6 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:6);    -   g) the GG or GT genotype for the Single Nucleotide Polymorphism        marker SNP_07 in SEQ ID NO: 7 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:7);    -   h) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_01 and SNP_07 (e.g. in-between SNP_01 and SNP_06, SNP_01 and        SNP_05, SNP_01 and SNP_04, SNP_01 and SNP_03, SNP_01 and        SNP_02); or in between SNP_02 and SNP_07 (e.g. in-between SNP_02        and SNP_06, SNP_02 and SNP_05, SNP_02 and SNP_04, SNP_02 and        SNP_03); or in between SNP_03 and SNP_07 (e.g. in-between SNP_03        and SNP_06, SNP_03 and SNP_05, SNP_03 and SNP_04); or in between        SNP_04 and SNP_7 (e.g. in-between SNP_04 and SNP_06, SNP_04 and        SNP_05); or in between SNP_05 and SNP_07 (e.g. in-between SNP_05        and SNP_06); or in between SNP_06 and SNP_07.

As mentioned, the skilled person can also develop other molecularmarkers, e.g. a wild L. virosa genome specific marker in-between markerSNP_01 and SNP_07 and/or within 7 cM or within 5 cM of any one of SNP_01to SNP_07, and/or within 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb,0.3 Mb, 0.2 Mb, 0.1 Mb, 50 kb, 20 kb, 10 kb, 5 kb or less of any one ofSNP_01 to SNP_07. Such markers may also be a stretch of nucleotide, CAPSmarkers, INDELs, etc. The skilled person can, for example, sequence theintrogression fragment or the QTL region and use the sequenceinformation to develop new markers and marker assays.

In another aspect the introgression fragment on chromosome 6 (comprisingQTL6.1 or a variant) is detectable by a molecular marker assay whichdetects at least 1, preferably at least 2 or 3, or at least 4, 5, 6, orall 7 of the markers selected from the group consisting of:

-   -   a) the AA or AT genotype for the Single Nucleotide Polymorphism        marker SNP_01 in SEQ ID NO: 1 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:1);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:2);    -   c) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:3);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_04 in SEQ ID NO: 4 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:4);    -   e) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:5);    -   f) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_06 in SEQ ID NO: 6 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:6);    -   g) the GG or GT genotype for the Single Nucleotide Polymorphism        marker SNP_07 in SEQ ID NO: 7 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:7).

In another aspect a cultivated L. saliva plant is provided comprising anintrogression fragment on chromosome 6 in homozygous or heterozygousform, wherein said introgression fragment confers Nr: 1 resistance andwherein said introgression fragment is detectable by a molecular markerassay which detects at least 2, 3 or 4 (or at least 5, 6 or all 7)consecutive markers selected from the group consisting of:

-   -   a) the AA or AT genotype for the Single Nucleotide Polymorphism        marker SNP_01 in SEQ ID NO: 1 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:1);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:2);    -   c) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:3);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_04 in SEQ ID NO: 4 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:4);    -   e) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:5);    -   f) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_06 in SEQ ID NO: 6 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:6);    -   g) the GG or GT genotype for the Single Nucleotide Polymorphism        marker SNP_07 in SEQ ID NO: 7 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:7).

The SNP markers SNP_01 to SNP_07 are located in the given order on theintrogression fragment. Consecutive markers refers to markers in thesame consecutive order, so e.g. two consecutive markers may be SNP_01and SNP_02; SNP_02 and SNP_03; SNP_03 and SNP_04, etc. and threeconsecutive markers may be SNP_01 and SNP_02 and SNP_03; SNP_02 andSNP_03 and SNP_04; etc.

The fragment may, thus, be smaller and lack 1, 2, 3, 4, 5 or even 6 ofthe markers, but it may still confer Nr:1 resistance on the cultivatedlettuce plant, i.e. it can still comprise the Nr:1 allele. Such smallerintrogression fragments are an embodiment of the invention.

Plants having smaller introgression fragments can be generated e.g. bystarting with a plant comprising a large introgression fragment andcrossing such a plant with another cultivated lettuce plant and selfingthe progeny of said cross to generate a population of plants which maycontain recombinants having a smaller introgression fragment onchromosome 6. Marker assays can be used to determine the size of thesmaller introgression fragment. One or more of SNP markers SNP_01 toSNP_07 may be missing (i.e. the plant may only comprise 1, 2, 3, 4, 5, 6of the SNP markers). The Nr:1 resistance phenotype of plants comprisingsuch a smaller introgression fragment can then be determined asdescribed herein, i.e. growing a plurality of plants comprising thesmaller introgression fragment in a controlled environment or fieldexperiments together with suitable control plants, lacking theintrogression fragment. The assay may be a free choice or non-choiceassay, as the resistance identified herein confers both types ofresistance. The control plants are preferably a genetic control or asusceptible variety, such as Mafalda. If the Nr:1 resistance remainssignificantly higher than in the control, then the smaller introgressionfragment has retained the QTL6.1 (or a variant thereof).

Alternatively, the same or variant QTL (QTL6.1 or variant QTL6.1) may beintrogressed from a different wild source, whereby optionally not allSNP markers disclosed herein may be present. Such alternative wildsources are preferably L. virosa accessions. They can be identifiedusing the SNP markers provided herein, by screening wild germplasm (e.g.L. virosa accessions) using a marker assay to detect the genotype ofmarkers SNP_01 to SNP_07 or any marker in-between SNP_01 and SNP_07.Alternatively such wild sources can be identified phenotypically andoptionally screened at a later stage for the presence of one or more ofthe markers described, or optionally progeny from crosses with suchaccessions can be screened for the markers. Plants comprising the sameor variant QTL6.1 from other sources are also an embodiment of theinvention. As long as at least 1, 2, 3, 4, 5, 6 or more of the SNPs,preferably at least 2, 3, 4, 5, 6 or more consecutive SNP markers ofSNP_01 to SNP_07 also have the SNP genotype indicative of the QTL, theplant comprises QTL6.1 (or a variant thereof). The skilled person canintrogress the QTL6.1 (or a variant thereof) into cultivated lettuce inorder confer Nr:1 resistance as described herein.

In a specific embodiment the plant of the invention comprises anintrogression fragment comprising at least a subset of SNP markers, i.e.at least 1, 2, 3, 4, or all 5 of the following markers selected from thegroup consisting of:

-   -   a) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:2);    -   b) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:3);    -   c) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_04 in SEQ ID NO: 4 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:4);    -   d) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:5);    -   e) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_06 in SEQ ID NO: 6 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:6); and optionally    -   f) any wild lettuce, especially L. virosa, genome-specific        marker between SNP_02 and SNP_06.

Preferably, the plant of the invention comprises an introgressionfragment comprising at least SNP markers SNP_03, SNP_04, SNP_05 and/orSNP_06 (or any marker in-between any of these), especially at leastSNP_04, SNP_05 and/or SNP_06 (or any marker in-between any of these).

Thus, the introgression fragment (and a cultivated lettuce plant orplant part, e.g., a cell, comprising the introgression fragment) can bedetected in a marker assay by detecting the SNP genotype of theintrogression fragment (i.e. of the wild lettuce, e.g. L. virosagermplasm) of one or more or all of the markers above.

In yet another aspect, the plant of the invention comprises anintrogression fragment comprising at least SNP_04, i.e. theintrogression fragment is detected in a marker assay detecting the GG orGA genotype for the Single Nucleotide Polymorphism marker SNP_04 in SEQID NO: 4. Optionally also the flanking markers, SNP_03 and/or SNP_05 aredetected, i.e. the introgression fragment is detected in a marker assaydetecting at least SNP_04 and optionally also at least one of thefollowing markers:

-   -   the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:5); and/or    -   the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:3); and optionally    -   any wild lettuce, especially L. virosa, genome-specific marker        between SNP_03 and SNP_05.        Lettuce Plants Comprising an Introgression Fragment on        Chromosome 6 (Comprising QTL 6.1 or a Variant Thereof)

Based on the later QTL mapping data the QTL region could be specifiedand cultivated lettuce plants comprising an introgression fragment fromLactuca virosa, wherein the introgression fragment comprises QTL6.1 (ora variant thereof) are provided, whereby the introgression fragmentcomprises all or part of the region starting at 77 Mb on chromosome 6and ending at 161 Mb on chromosome 6.

Thus, in one aspect a Lactuca sativa plant comprising an introgressionfragment from Lactuca virosa on chromosome 6 is provided which comprisesa Quantitative Trait Locus that confers resistance against Nasonoviaribisnigri biotype 1 (Nr:1), and wherein the introgression fragment onchromosome 6 comprises all or part of the region starting at 77 Mb andending at 161 Mb of chromosome 6.

It is understood that a smaller introgression fragment (i.e. comprisinga resistance conferring part of the above mentioned region spanning 77Mb to 161 Mb of chromosome 6) which retains the QTL6.1 (or variant) maybe a fragment having a size of 80 Mb, 70 Mb, 60 Mb, 50 Mb, 40 Mb, 30 Mb,20 Mb, 10 Mb, 5 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 100 kb, 50 kb or lessand comprise the QTL6.1 or a variant thereof. In one aspect the part isat least 5 kb, 10 kb, 20 kb in size, or more.

In one aspect, the introgression fragment on chromosome 6 is comprisesand is detectable by a molecular marker assay which detects at leastone, preferably at least 2 or 3 or 4 or 5 (or more) of the markersselected from the group consisting of:

-   -   a) The CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP1.23 in SEQ ID NO: 23 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 23);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 2);    -   c) the TT or CT genotype for the Single Nucleotide Polymorphism        marker SNP2.24 in SEQ ID NO: 24 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 24);    -   the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 3);    -   d) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP1.23 and SNP_03 (e.g. in-between SNP1.23 and SNP2.24, SNP1.23        and SNP_02); or in between SNP_02 and SNP_03 (e.g. in-between        SNP_02 and SNP2.24); or in between SNP2.24 and SNP_03;    -   e) any wild lettuce genome specific marker especially L.        virosa-genome specific marker, located within a distance of 10        Mb, preferably within 5 Mb, of any marker selected from SNP1.23,        SNP_02, SNP2.24, or SNP_03.

Optionally, in one aspect, the introgression fragment comprises (and isdetectable by) a L. virosa accession specific marker selected from theGG or GT genotype for the Single Nucleotide Polymorphism marker VSP1 inSEQ ID NO: 26 (or in a sequence comprising substantial sequence identityto SEQ ID NO: 26) and the AA or AC genotype for the Single NucleotidePolymorphism marker VSP3 in SEQ ID NO: 27 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 27). Using the SNP markersVSP1 and VSP3 the introgression fragments comprising QTL6.1 fromdifferent L. virosa type accessions can be distinguished.

The introgression fragment may be in heterozygous or homozygous form, asindicated by the SNP genotype. So in one aspect the introgressionfragment is in homozygous form and the SNP marker genotype is thehomozygous genotype.

As mentioned, variants of QTL6.1 may be identified and introgressed fromvarious Nr:1 resistant Lactuca virosa accessions. Such variants maycomprise a genomic sequence which is not 100% identical to the sequencesprovided herein, but may still have substantial sequence identity (suchas at least 85%, 90% or more) when genomic sequences of the same lengthsare aligned. That there is variation in the QTL region where QTL6.1 islocated can be seen due to the fact that accession specific SNP markerscould be identified in introgressions of QTL6.1 from two different L.virosa accessions, which introgressions however both comprise theresistance conferring QTL6.1. So the introgression fragment comprisingVSP1 is a different introgression fragment than the one comprising VSP3,but both comprise QTL6.1. Within wild L. virosa accessions, whichcomprise QTL6.1, there may, thus, be genomic variation in the regionspanning 77 Mb to 161 Mb on chromosome 6. However, such accessions mayequally be used to introgress all or part of the region starting at 77Mb and ending at 161 Mb of chromosome 6 into Nr: 1 susceptiblecultivated lettuce, in order to generate plants of the invention. Withthe knowledge of the instant invention, that the region comprises a QTL,the skilled person can introgress the same region or a smallerresistance conferring part into cultivated lettuce.

In one aspect the introgression fragment on chromosome 6 is comprises,and is detectable by a molecular marker assay which detects, at leastone, preferably at least 2 or 3 or 4 of the markers selected from thegroup consisting of:

-   -   a) The CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP1.23 in SEQ ID NO: 23 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 23;    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 2;    -   c) the TT or CT genotype for the Single Nucleotide Polymorphism        marker SNP2.24 in SEQ ID NO: 24 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 24;    -   d) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 3.

In one aspect the introgression fragment is derivable from seedsdeposited under NCIMB42086 or progeny thereof.

In one aspect the introgression fragment is from another Nr:1 resistantL. virosa accession, such as an accession comprising the AA or ACgenotype for the Single Nucleotide Polymorphism marker VSP3 in SEQ IDNO: 27 (or in a sequence comprising substantial sequence identity to SEQID NO: 27).

In another aspect the introgression fragment is from another Nr:1resistant L. virosa accession, such as an accession comprising the GG orGT genotype for the Single Nucleotide Polymorphism marker VSP1 in SEQ IDNO: 26 (or in a sequence comprising substantial sequence identity to SEQID NO: 26).

Other aspects, described elsewhere based on the first QTL analysis andmarkers SNP_01 to SNP_07 equally apply to the markers and introgressionidentified in this later analysis. So, for example, QTL6.1 can beintrogressed into any cultivated lettuce, especially Nr:1 susceptiblelines or varieties by e.g. backcrossing. It can also be combined incultivated lettuce with a recombinant chromosome 7, comprising QTL7.1and/or QTL7.2.

Lettuce Plants Comprising an Introgression Fragment on Chromosome 7 (QTL7.1 and/or QTL7.2 or Variants of these)

Based on the first QTL mapping results, the following cultivated lettuceplants are encompassed herein.

In one aspect the introgression fragment comprising QTL 7.2 or a variantthereof (and the cultivated lettuce plant or plant part comprising theintrogression fragment) on chromosome 7 is detectable by a molecularmarker assay which detects at least 1, preferably at least 2 or 3, or atleast 4, 5, 6, or 7 of the markers selected from the group consistingof:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:9);    -   c) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:10);    -   d) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 11);    -   e) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_12 in SEQ ID NO: 12 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:12);    -   f) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_13 in SEQ ID NO: 13 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:13);    -   g) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 14 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:14);    -   h) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_08 and SNP_14 (e.g. in-between SNP_08 and SNP_13, SNP_08 and        SNP_12, SNP_08 and SNP_11, SNP_08 and SNP_10, SNP_08 and        SNP_09); or in between SNP_09 and SNP_14 (e.g. in-between SNP_09        and SNP_13, SNP_09 and SNP_12, SNP_09 and SNP_11, SNP_09 and        SNP_10); or in between SNP_10 and SNP_14 (e.g. in-between SNP_10        and SNP_13, SNP_10 and SNP_12, SNP_10 and SNP_11); or in between        SNP_11 and SNP_14 (e.g. in-between SNP_11 and SNP_13, SNP_11 and        SNP_12); or in between SNP_12 and SNP_14 (e.g. in-between SNP_12        and SNP_13); or in between SNP_13 and SNP_14.

As mentioned, the skilled person can also develop other molecularmarkers, e.g. a wild lettuce genome specific marker, e.g. L. virosagenome-specific markers in between marker SNP_08 and SNP_14 and/orwithin 7 cM or within 5 cM of any one of SNP_08 to SNP_14, and/or within5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb,50 kb, 20 kb, 10 kb, 5 kb or less of any one of SNP_08 to SNP_14. Suchmarkers may also be a stretch of nucleotide, CAPS markers, INDELs, etc.

In another aspect the introgression fragment on chromosome 7 (comprisingQTL 7.2 or a variant) is detectable by a molecular marker assay whichdetects at least 1, preferably at least 2 or 3, or at least 4, 5, 6, orall 7 of the markers selected from the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:9);    -   c) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:10);    -   d) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 11);    -   e) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_12 in SEQ ID NO: 12 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:12);    -   f) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_13 in SEQ ID NO: 13 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:13);    -   g) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 14 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 14).

In another aspect a cultivated lettuce plant is provided comprising anintrogression fragment on chromosome 7 in homozygous or heterozygousform, wherein said introgression fragment comprises QTL7.2 conferringNr:1 resistance and wherein said introgression fragment is detectable bya molecular marker assay which detects at least 2, 3 or 4 (or at least5, 6, 7) consecutive markers selected from the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:9);    -   c) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:10);    -   d) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 11);    -   e) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_12 in SEQ ID NO: 12 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:12);    -   f) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_13 in SEQ ID NO: 13 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:13);    -   g) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 14 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:14).

The SNP markers SNP_08 to SNP_14 are located in the given order on theintrogression fragment. Consecutive markers refers to markers in thesame consecutive order, so e.g. two consecutive markers may be SNP_08and SNP_09; SNP_09 and SNP_10; SNP_10 and SNP_11, etc. and threeconsecutive markers may be SNP_08 and SNP_09 and SNP_10; SNP_09 andSNP_10 and SNP_11; etc.

The fragment may, thus, be smaller and lack 1, 2, 3, 4, 5, 6 of themarkers, but it may still confer Nr:1 resistance on the cultivatedlettuce plant, i.e. it can still comprise the Nr:1 allele. Such smallerintrogression fragments are an embodiment of the invention. Plantshaving smaller introgression fragments can be generated e.g. by startingwith a plant comprising a large introgression fragment and crossing sucha plant with another cultivated lettuce plant and selfing the progeny ofsaid cross to generate a population of plants which may containrecombinants having a smaller introgression fragment on chromosome 7.Marker assays can be used to determine the size of the smallerintrogression fragment. One or more of SNP markers SNP_08 to SNP_14 maybe missing (i.e. the plant may only comprise 1, 2, 3, 4, 5, or 6 of theSNP markers). The Nr:1 resistance of plants comprising such a smallerintrogression fragment can then be compared in Nr: 1 assays as describedherein, i.e. growing a plurality of plants comprising the smallerintrogression fragment in field experiments together with suitablecontrol plants, lacking the introgression fragment. The control plantsare preferably a genetic control or a susceptible control such asMafalda. If the Nr:1 resistance remains significantly higher than in thecontrol, then the smaller introgression fragment has retained the QTL7.2(or variant).

Alternatively, the same or variant QTL (QTL7.2 or variant QTL7.2) may beintrogressed from a different wild source, such as different L. virosaaccessions, whereby optionally not all SNP markers disclosed herein maybe present. Such alternative wild sources can be identified using theSNP markers provided herein, by screening wild germplasm, e.g. L. virosaaccessions using a marker assay to detect the genotype of markers SNP_08to SNP_14, or a marker in between these. Alternatively such wild sourcescan be identified phenotypically and optionally screened at a laterstage for the presence of one or more of the markers described, oroptionally progeny from crosses with such accessions can be screened forthe markers. Plants comprising the QTL7.2 or variant QTL7.2 from othersources are also an embodiment of the invention. As long as at least 1,2, 3, 4, 5, 6, or 7 or more of the SNPs, preferably at least 2, 3, 4, 5,6, or 7 consecutive SNP markers of SNP_08 to SNP_14 also have SNPgenotype indicative of the QTL, the plant comprises QTL7.2 (or a variantthereof). The skilled person can introgress the QTL7.2 (or a variantthereof) into cultivated lettuce in order to generate Nr: 1 resistanceas described herein.

In a specific embodiment the plant of the invention comprises anintrogression fragment comprising at least a subset of SNP markers, i.e.at least 1, 2, 3, 4 or all 5 of the following markers selected from thegroup consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:9);    -   c) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:10);    -   d) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 11);    -   e) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_12 in SEQ ID NO: 12 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:12); and optionally    -   f) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_08 and SNP_12.

Especially, in one aspect the cultivated lettuce plant of the inventioncomprises at least 1, 2 or 3 markers selected from the group consistingof:

-   -   a) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:9);    -   b) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:10);    -   c) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:11); and optionally    -   d) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_09 and SNP_11.

Thus, the introgression fragment (and a cultivated lettuce plant orplant part, e.g., a cell, comprising the introgression fragment) can bedetected in a marker assay by detecting the SNP genotype of theintrogression fragment (i.e. of the wild lettuce germplasm) of one ormore or all of the markers above.

In one aspect the introgression fragment comprising QTL 7.1 (and thecultivated lettuce plant or plant part comprising the introgressionfragment) on chromosome 7 is detectable by a molecular marker assaywhich detects at least 1, preferably at least 2 or 3, or at least 4, 5,6, 7 or 8 of the markers selected from the group consisting of:

-   -   a) the TT or TA genotype for the Single Nucleotide Polymorphism        marker SNP_15 in SEQ ID NO: 15 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 15);    -   b) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_16 in SEQ ID NO: 16 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:16);    -   c) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:17);    -   d) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:18);    -   e) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:19);    -   f) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:20);    -   g) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_21 in SEQ ID NO: 21 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:21);    -   h) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_22 in SEQ ID NO: 22 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:22);    -   i) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_15 and SNP_22 (e.g. in-between SNP_15 and SNP_21, SNP_15 and        SNP_20, SNP_15 and SNP_19, SNP_15 and SNP_18, SNP_15 and SNP_17,        SNP_15 and SNP_16); or in between SNP_16 and SNP_22 (e.g.        in-between SNP_16 and SNP_21, SNP_16 and SNP_20, SNP_16 and        SNP_19, SNP_16 and SNP_18, SNP_16 and SNP_17); or in between        SNP_17 and SNP_22 (e.g. in-between SNP_17 and SNP_21, SNP_17 and        SNP_20, SNP_17 and SNP_19, SNP_17 and SNP_18); or in between        SNP_18 and SNP_22 (e.g. in-between SNP_18 and SNP_21, SNP_18 and        SNP_20, SNP_18 and SNP_19); or in between SNP_19 and SNP_22        (e.g. in-between SNP_19 and SNP_21, SNP_19 and SNP_20); or in        between SNP_20 and SNP_22; or in between SNP_21 and SNP_22.

As mentioned, the skilled person can also develop other molecularmarkers, e.g. a wild lettuce genome specific marker, e.g. L. virosagenome-specific markers, in between marker SNP_15 and SNP_22 and/orwithin 7 cM or within 5 cM of any one of SNP_15 to SNP_22, and/or within5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb,50 kb, 20 kb, 10 kb, 5 kb or less of any one of SNP_15 to SNP_22. Suchmarkers may also be a stretch of nucleotide, CAPS markers, INDELs, etc.

In another aspect the introgression fragment on chromosome 7 (comprisingQTL 7.1 or a variant) is detectable by a molecular marker assay whichdetects at least 1, preferably at least 2 or 3, or at least 4, 5, 6, 7or all 8 of the markers selected from the group consisting of:

-   -   a) the TT or TA genotype for the Single Nucleotide Polymorphism        marker SNP_15 in SEQ ID NO: 15 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:15);    -   b) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_16 in SEQ ID NO: 16 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:16);    -   c) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:17);    -   d) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:18);    -   e) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:19);    -   f) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:20);    -   g) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_21 in SEQ ID NO: 21 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:21);    -   h) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_22 in SEQ ID NO: 22 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:22).

In another aspect a cultivated lettuce plant is provided comprising anintrogression fragment on chromosome 7 in homozygous or heterozygousform, wherein said introgression fragment comprises QTL7.1 conferringNr: 1 resistance and wherein said introgression fragment is detectableby a molecular marker assay which detects at least 2, 3 or 4 (or atleast 5, 6, 7, 8) consecutive markers selected from the group consistingof:

-   -   a) the TT or TA genotype for the Single Nucleotide Polymorphism        marker SNP_15 in SEQ ID NO: 15 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:15);    -   b) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_16 in SEQ ID NO: 16 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:16);    -   c) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:17);    -   d) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:18);    -   e) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:19);    -   f) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:20);    -   g) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_21 in SEQ ID NO: 21 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:21);    -   h) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_22 in SEQ ID NO: 22 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:22).

The SNP markers SNP_15 to SNP_22 are located in the given order on theintrogression fragment. Consecutive markers refers to markers in thesame consecutive order, so e.g. two consecutive markers may be SNP_12and SNP_13; SNP_13 and SNP_14; SNP_14 and SNP_15, etc. and threeconsecutive markers may be SNP_12 and SNP_13 and SNP_14; SNP_13 andSNP_14 and SNP_15; etc.

The fragment may, thus, be smaller and lack 1, 2, 3, 4, 5, 6 or 7 of themarkers, but it may still confer Nr:1 resistance on the cultivatedlettuce plant, i.e. it can still comprise the Nr:1 allele. Such smallerintrogression fragments are an embodiment of the invention. Plantshaving smaller introgression fragments can be generated e.g. by startingwith a plant comprising a large introgression fragment and crossing sucha plant with another cultivated lettuce plant and selfing the progeny ofsaid cross to generate a population of plants which may containrecombinants having a smaller introgression fragment on chromosome 7(comprising QTL 7.1 or a variant). Marker assays can be used todetermine the size of the smaller introgression fragment. One or more ofSNP markers SNP_15 to SNP_22 may be missing (i.e. the plant may onlycomprise 1, 2, 3, 4, 5, 6 or 7 of the SNP markers). The Nr:1 resistanceof plants comprising such a smaller introgression fragment can then becompared in Nr:1 assays as described herein, i.e. growing a plurality ofplants comprising the smaller introgression fragment in fieldexperiments together with suitable control plants, lacking theintrogression fragment. The control plants are preferably a geneticcontrol or a susceptible control such as Mafalda. If the Nr: 1resistance remains significantly higher than in the control, then thesmaller introgression fragment has retained the QTL7.1 (or a variant).

Alternatively, the same or variant QTL (QTL7.1 or variant QTL7.1) may beintrogressed from a different wild source, such as different L. virosaaccessions, whereby optionally not all SNP markers disclosed herein maybe present. Such alternative wild sources can be identified using theSNP markers provided herein, by screening wild germplasm, e.g. L. virosaaccessions using a marker assay to detect the genotype of markers SNP_15to SNP_22, or a marker in-between these. Alternatively such wild sourcescan be identified phenotypically and optionally screened at a laterstage for the presence of one or more of the markers described, oroptionally progeny from crosses with such accessions can be screened forthe markers. Plants comprising the QTL7.1 or variant QTL7.1 from othersources are also an embodiment of the invention. As long as at least 1,2, 3, 4, 5, 6, 7 or 8 or more of the SNPs, preferably at least 2, 3, 4,5, 6, 7 or 8 consecutive SNP markers of SNP_15 to SNP_22 also have SNPgenotype indicative of the QTL, the plant comprises QTL7.1 (or a variantthereof). The skilled person can introgress the QTL7.1 (or a variantthereof) into cultivated lettuce in order to generate Nr:1 resistance asdescribed herein.

In a specific embodiment the plant of the invention comprises anintrogression fragment comprising at least a subset of SNP markers, i.e.at least 1, 2, 3, 4 or all 5 of the following markers selected from thegroup consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:17);    -   b) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18);    -   c) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:19);    -   d) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:20);    -   e) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_21 in SEQ ID NO: 21 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:21); and optionally    -   f) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_19 and SNP_21.

Especially, in one aspect the cultivated lettuce plant of the inventioncomprises at least 1, 2 or 3 markers selected from the group consistingof:

-   -   a) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:19);    -   b) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:20);    -   c) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_21 in SEQ ID NO: 21 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:21); and optionally    -   d) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_19 and SNP_21.

Thus, the introgression fragment (and a cultivated lettuce plant orplant part, e.g., a cell, comprising the introgression fragment) can bedetected in a marker assay by detecting the SNP genotype of theintrogression fragment (i.e. of the wild lettuce germplasm) of one ormore or all of the markers above.

Lettuce Plants Comprising an Introgression Fragment on Chromosome 7 (QTL7.1 or a Variant Thereof)

Based on the later QTL mapping data the QTL7.1 region could be specifiedand cultivated lettuce plants comprising an introgression fragment fromLactuca virosa, wherein the introgression fragment comprises QTL7.1 (ora variant thereof) are provided, whereby the introgression fragmentcomprises all or part of the region starting at 203 Mb on chromosome 7and ending at 219 Mb on chromosome 7.

Thus, in one aspect a Lactuca sativa plant comprising an introgressionfragment from Lactuca virosa on chromosome 7 is provided which comprisesa Quantitative Trait Locus that confers resistance against Nasonoviaribisnigri biotype 1 (Nr:1), and wherein the introgression fragment onchromosome 7 comprises all or part of the region starting at 203 Mb onchromosome 7 and ending at 219 Mb of chromosome 7.

It is understood that a smaller introgression fragment (i.e. comprisinga resistance conferring part of the above mentioned region spanning 203Mb to 219 Mb of chromosome 7) which retains the QTL7.1 (or variant) maybe a fragment having a size of 15 Mb, 10 Mb, 5 Mb, 2.5 Mb, 2 Mb, 1 Mb,0.5 Mb, 100 kb, 50 kb or less and comprise the QTL7.1 or a variantthereof. In one aspect the part is at least 5 kb, 10 kb, 20 kb in size,or more.

In one aspect, the introgression fragment on chromosome 7 is comprisesand is detectable by a molecular marker assay which detects at leastone, preferably at least 2 or 3 or 4 or 5 (or more) of the markersselected from the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 17);    -   b) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP17.25 in SEQ ID NO: 25 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 25);    -   c) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19);    -   e) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_17 and SNP_19 (e.g. in-between SNP_17 and SNP_18, in between        SNP_17 and SNP17.25; or in between SNP17.25 and SNP_19, or in        between SNP17.25 and SNP_18, or in between SNP_18 and SNP_19);    -   f) any wild lettuce genome specific marker especially L.        virosa-genome specific marker, located within a distance of 12        Mb or of 10 Mb, preferably within 5 Mb, of any marker selected        from SNP_17, SNP_17.25, SNP_18 and SNP_19.

Optionally, in one aspect, the introgression fragment comprises (and isdetectable by) a L. virosa accession specific marker selected from theCC or AC genotype for the Single Nucleotide Polymorphism marker VSP2 inSEQ ID NO: 28 (or in a sequence comprising substantial sequence identityto SEQ ID NO: 28) and the GG or GA genotype for the Single NucleotidePolymorphism marker VSP4 in SEQ ID NO: 29 (or in a sequence comprisingsubstantial sequence identity to SEQ ID NO: 29). Using the SNP markersVSP2 and VSP4 the introgression fragments comprising QTL7.1 from twodifferent types L. virosa accessions can be distinguished.

The introgression fragment may be in heterozygous or homozygous form, asindicated by the SNP genotype. So in one aspect the introgressionfragment is in homozygous form and the SNP marker genotype is thehomozygous genotype.

As mentioned, variants of QTL7.1 may be identified and introgressed fromvarious Nr:1 resistant Lactuca virosa accessions. Such variants maycomprise a genomic sequence which is not 100% identical to the sequencesprovided herein, but may still have substantial sequence identity (suchas at least 85%, 900 or more) when genomic sequences of the same lengthsare aligned. That there is variation in the QTL region where QTL7.1 islocated can be seen due to the fact that accession specific SNP markerscould be identified in introgressions of QTL7.1 from two different L.virosa accessions, which introgressions however both comprise theresistance conferring QTL7.1. So the introgression fragment comprisingVSP2 is a different introgression fragment than the one comprising VSP4,but both comprise QTL7.1. Within wild L. virosa accessions, whichcomprise QTL7.1, there may, thus, be genomic variation in the regionspanning 203 Mb to 219 Mb on chromosome 7. However, such accessions mayequally be used to introgress all or part of the region starting at 203Mb and ending at 219 Mb of chromosome 7 into Nr: 1 susceptiblecultivated lettuce, in order to generate plants of the invention. Withthe knowledge of the instant invention, that the region comprises a QTL,the skilled person can introgress the same region or a smallerresistance conferring part into cultivated lettuce.

In one aspect the introgression fragment on chromosome 7 is comprises,and is detectable by a molecular marker assay which detects, at leastone, preferably at least 2 or 3 or 4 of the markers selected from thegroup consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 17);    -   b) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP17.25 in SEQ ID NO: 25 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 25);    -   c) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19);

In one aspect the introgression fragment is derivable from seedsdeposited under NCIMB42086 or progeny thereof.

In one aspect the introgression fragment is from another Nr:1 resistantL. virosa accession, such as an accession comprising the GG or GAgenotype for the Single Nucleotide Polymorphism marker VSP4 in SEQ IDNO: 29 (or in a sequence comprising substantial sequence identity to SEQID NO:29).

In another aspect the introgression fragment is from another Nr:1resistant L. virosa accession, such as an accession comprising the CC orCA genotype for the Single Nucleotide Polymorphism marker VSP2 in SEQ IDNO: 28 (or in a sequence comprising substantial sequence identity to SEQID NO:28).

Other aspects, described elsewhere based on the first QTL analysis ofQTL7.1 and markers SNP_15 to SNP_22 equally apply to the markers andintrogression identified in this later analysis. So, for example, QTL7.1can be introgressed into any cultivated lettuce, especially Nr:1susceptible lines or varieties by e.g. backcrossing. It can also becombined in cultivated lettuce with a recombinant chromosome 6comprising QTL6.1 and/or with a second QTL on chromosome 7, namelyQTL7.2.

Thus, in one aspect three Quantitative Trait Loci (QTL6.1 and/or QTL7.1and/or QTL7.2) were found to be present on chromosome 6 and 7 of wildlettuce (especially L. virosa, such as accession NCIMB42086) which, whentransferred (introgressed) into a cultivated lettuce variety or breedingline separately or in combination, and when present in heterozygous orhomozygous form, confer Nr:1 resistance onto the cultivated lettuceplant. In one aspect the cultivated lettuce plant comprises theintrogression fragment(s) on only one of the chromosome 6 and/or 7,while the homologous chromosomes 6 and 7 of the pair may be anon-recombinant chromosome 6 and/or 7 of cultivate lettuce lacking theintrogression fragment(s). In another aspect the cultivated lettuceplant comprises the introgression fragment(s) on both of the chromosome6 and/or 7 of the homologous pair (the introgression is in homozygousform). Genotypes of the cultivated lettuce plants may thus be for plantscomprising a single introgression fragment: QTL6.1/wt, QTL6.1/QTL6.1,QTL7.1/wt, QTL7.1/QTL7.1, QTL7.2/wt, QTL7.2/QTL7.2; for plantscomprising two introgression fragments, one on chromosome 6 and one onchromosome 7: QTL6.1/wt plus QTL7.1/wt, QTL6.1/QTL6.1 plus QTL7.1/wt,QTL6.1/QTL6.1 plus QTL7.1/QTL7.1, QTL6.1/wt plus QTL7.1/QTL7.1;QTL6.1/wt plus QTL7.2/wt, QTL6.1/QTL6.1 plus QTL7.2/wt, QTL6.1/QTL6.1plus QTL7.2/QTL7.2, QTL6.1/wt plus QTL7.2/QTL7.2; for plants comprisingtwo introgression fragments on chromosome 7: QTL7.2/wt plus QTL7.1/wt,QTL7.2/QTL7.2 plus QTL7.1/wt, QTL7.2/QTL7.2 plus QTL7.1/QTL7.1,QTL7.2/wt plus QTL7.1/QTL7.1. The plants comprising two introgressionfragments, one on chromosome 6 and one on chromosome 7, as describedabove, may additionally comprise the third QTL on chromosome 7 inheterozygous or homozygous form.

The introgression fragments may be from the same accession, but they mayalso be from different accessions. In one aspect, the introgressionfragments on chromosome 6 are from the same accession as theintrogressions fragments on chromosome 7. However, one can also combineintrogression fragments from different accessions, e.g. those onchromosome 6 may be from one accession and those on chromosome 7 may befrom another accession. E.g. markers VSP1 and VSP2 are from oneaccession, while markers VSP3 and VSP4 are from a different accession.In one aspect the introgression fragment on chromosome 6 comprises theVSP1 marker and the introgession fragment on chromosome 7 comprises theVSP2 marker. In another aspect the introgression fragment on chromosome6 comprises the VSP3 marker and the introgression fragment on chromosome7 comprises the VSP4 marker. But the introgression fragment onchromosome 6 and 7 may also be from different accessions, so e.g. thaton chromosome 6 may comprises VSP1, while that on chromosome 7 maycomprise VSP4, or the fragment on chromosome 6 may comprise the VSP3marker and that on chromosome 7 the VSP2 marker. Likewise, a planthomozygous for an introgression fragment, e.g. comprising QTL6.1, maycontain the same fragment in homozygous form, but may also contain twodifferent introgression fragments.

Although the present sources of the three QTLs are specific wildsources, there are likely other wild lettuce accessions (especially L.virosa accessions) which comprise QTL6.1 and/or QTL7.1 and/or QTL7.2 atthe same locus/loci on chromosome 6 and/or 7. Such loci may compriseNr:1 alleles which have slightly different nucleotide sequences, i.e.variants of the alleles (QTLs) found herein. Such variant QTLs can alsobe identified and introgressed into cultivated lettuce as describedherein, to generate a cultivated lettuce plant comprising a genome ofcultivated L. sativa and a recombinant chromosome 6 and/or 7, wherebythe recombinant chromosome 6 and/or 7 comprises a wild Lactuca speciesintrogression fragment (especially L. virosa), which confers Nr: 1resistance onto the cultivated lettuce plant when present in homozygousor heterozygous form.

To identify such wild lettuce plants comprising QTL6.1 and/or QTL7.1and/or QTL7.2 (or variant QTLs), wild accessions can be screened, e.g.in a marker assay or by sequence comparison or other methods, for thepresence of one or more of the SNP markers provided herein. The putativeNr:1 resistance conferring QTLs (or variant QTLs) can then beintrogressed into cultivated lettuce, e.g. optionally using MAS (markerassisted selection), i.e. using one or more (or all) of the SNP markersprovided herein (or markers in between these) to detect and/or selectprogeny plants (e.g. backcross plants) comprising a recombinantchromosome 6 and/or 7. The selected plants, i.e. the cultivated lettuceplants comprising an introgression fragment on chromosome 6 and/or 7wherein the introgression fragment on chromosome 6 is detectable by oneor more of the SNP markers SNP_01 to SNP_07 or alternatively one or moreof SNP markers SNP1.23, SNP_02, SNP2.24 or SNP_03, or markers in betweenany of these, (as described elsewhere herein), and/or wherein theintrogression fragment on chromosome 7 is detectable by one or more ofthe SNP markers SNP_08 to SNP_14, or markers in between these, (asdescribed elsewhere herein) detecting QTL7.2 or a variant thereof and/orone or more of the SNP markers SNP_15 to SNP_22 or alternatively SNP_17,SNP17.25, SNP_18 or SNP_19, or markers in between any of these, (asdescribed elsewhere herein) detecting QTL7.1 or a variant thereof, canthen be phenotyped for Nr:1 resistance together with the suitablecontrol plants, preferably at least the genetic control and/or a Nr:1susceptible plant such as Mafalda, in order to determine whether theintrogression fragment indeed confers Nr:1 resistance. One or more Nr:1resistance assays as described can be used.

Accessions of wild lettuce, such as L. virosa, are obtainable from theUSDA National Plant Germplasm System collection or other seedcollections, such as the CGN in Wageningen, and can thus be screened forthe presence of QTL6.1 (or a variant) and/or QTL7.1 (or a variant)and/or QTL7.2 (or a variant) using e.g. a marker assay as describedherein, and accessions comprising one or more of the SNP markersindicative of QTL 6.1 or a variant; and/or comprising one or more of theSNP markers indicative of QTL7.2 or variant; and/or comprising one ormore of the SNP markers indicative of QTL7.1 or variant can be crossedwith a cultivated lettuce plant having normal wild-type, non-recombinantchromosomes 6 and 7. The F2 generation (or further generation, such asthe F3 or preferably a backcross generation such as the BC1, BC2, BC3 orBC1S1, etc.) can then be screened for recombinant plants having theintrogression fragment or a resistance-conferring part thereof, usingthe molecular marker assays described herein. Alternatively, wildaccessions can be screened phenotypically using a Nr:1 resistance assayand only progeny plants obtained from crosses with such wild accessionsmay be screened for the presence of the markers (and introgressionfragments). Such progeny plants also fall within the scope of theinvention.

In a specific embodiment, the introgression fragment comprising the Nr:1resistance conferring QTL6.1 and/or the Nr:1 resistance conferringQTL7.1 and/or Nr:1 resistance conferring QTL7.2 is derivable from (orderived from) or obtainable from (or obtained from; or as present in)seeds, a representative sample of which has been deposited underaccession number NCIMB42086, or from progeny thereof. The progeny may beany progeny which retain the one or more (or all) SNP markers indicativeof the QTLs, as described. Thus, progeny are not limited to F1 or F2progeny of the deposit, but can be any progeny, whether obtained byselfing and/or by crossing with another lettuce plant.

In one embodiment the introgression fragment is identifiable by one ormore of the markers described elsewhere herein, especially markersSNP_01 to SNP_07 (or any marker in between these) for the introgressionfragment on chromosome 6 (QTL6.1 or variant) or alternatively SNP1.23,SNP_02, SNP2.24 and/or SNP_03 (or any marker in between these),optionally also VSP1 or VSP3; and SNP_08 to SNP_14 (or any marker inbetween these) for the introgression fragment on chromosome 7 referredto as QTL7.2 (or variant) and SNP_15 to SNP_22 (or any marker in betweenthese) or alternatively SNP_17, SNP17.25, SNP_18 and/or SNP_19 (or anymarker in between these), optionally VSP2 or VSP4, for the introgressionfragment on chromosome 7 referred to as QTL7.1 (or variant).

In one aspect the invention provides a cultivated lettuce plant line orvariety, having a genome of L. sativa which line or variety comprisesNr:1 resistance, wherein the Nr:1 resistance is conferred by anintrogression fragment on the cultivated lettuce chromosome 6 and/orchromosome 7, wherein said introgression fragment is obtained by (orobtainable by) crossing a Nr: 1 resistant L. virosa plant (whichcomprises one or more the markers disclosed herein linked to the QTLs)with a cultivated lettuce plant.

In a further aspect the invention provides a cultivated lettuce plantline or variety, having a genome of L. sativa which line or varietycomprises Nr:1 resistance, wherein the Nr:1 resistance is conferred byan introgression fragment on the cultivated lettuce chromosome 6 and/orchromosome 7, wherein said introgression fragment is obtained by (orobtainable by) crossing a plant grown from seeds deposited under NCIMB42086 or progeny of this plant (which comprises one or more the markersdisclosed herein linked to the QTLs) with a cultivated lettuce plant.

In yet another embodiment the invention relates to a plant of theinvention i.e. a cultivated L. sativa plant comprising an introgressionfragment from a wild lettuce on chromosome 6 and/or 7 in homozygous orheterozygous form and wherein said introgression fragment is a variantof the genomic sequence comprising the QTL(s) as found in seedsdeposited under number NCIMB 42086, i.e. it comprises the Nr:1 QTL(s),but the genomic sequence may be different. As wild accessions will begenetically divergent, the genomic sequence of an introgression fragmentcomprising QTL6.1 or QTL7.1 or QTL7.2 (these QTLs are herein alsoreferred to as variants or orthologs of QTL6.1, QTL7.1 and QTL7.2) fromother wild lettuce accessions (e.g. other L. virosa accessions than theone deposited under accession number NCIMB42086) will most likely not beidentical to the genomic sequence, and even the Nr: 1 resistanceconferring gene (comprising a promoter, introns and exons) may bedivergent in nucleotide sequence, but the function can be the same, i.e.conferring Nr: 1 resistance. The divergence can be seen in that certainSNP markers linked to (variant) QTL6.1 and/or (variant) QTL7.1 and/or(variant) QTL7.2 may be commonly found in various accessions, whileother SNP markers may only be found in specific accessions. So forexample not all of SNP_01 to SNP_7, or not all of SNP1.23, SNP_02,SNP2.24 or SNP_03, and/or SNP_08 to SNP_14 and/or SNP_15 to SNP_22, orSNP_17, SNP17.25, SNP_18 or SNP_19, may be found in other Nr:1 resistantwild lettuces (e.g. L. virosa accessions), while these accessions maystill comprise QTL variants in the same region. Likewise, the genomicsequence comprising each of the SNP markers may not be 100% identical tothe sequence provided herein, but may only have a sequence identity of(at least) 85%, 900, 95%, 98%, or 99%/0 to sequence provided herein,i.e. to any one of SEQ ID NO: 1 to SEQ ID NO: 22 and SEQ ID NO: 23 to29. However, the Nr: 1 resistance conferring QTL6.1 (or variant) andQTL7.1 (or variant) and QTL7.2 (or variant, comprising e.g. a variant orortholog of the Nr:1 allele) may still be present in such wildaccessions. The skilled person is capable of identifying andintrogressing the (variant) QTLs 6.1 and/or (variant) QTL 7.1 and/or(variant) QTL7.2 comprising region found in other wild lettuceaccessions (especially L. virosa accessions; in particular L. virosaaccessions comprising both free-choice and non-choice resistance againstNr: 1) into cultivated lettuce without undue burden.

In one embodiment the presence of the introgression fragment, or thechromosome 6 region (or variant or orthologous chromosome 6 region),comprising QTL6.1 (or variant), is detectable by a molecular markerassay which detects at least 1, preferably at least 2, 3, 4, 5, 6, ormore (or all 7) Single Nucleotide Polymorphism (SNP) markers selectedfrom the group consisting of:

-   -   a) the AA or AT genotype for the Single Nucleotide Polymorphism        marker SNP_01 in SEQ ID NO: 1 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:1);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:2);    -   c) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:3);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_04 in SEQ ID NO: 4 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:4);    -   e) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:5);    -   f) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_06 in SEQ ID NO: 6 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:6);    -   g) the GG or GT genotype for the Single Nucleotide Polymorphism        marker SNP_07 in SEQ ID NO: 7 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:7);    -   h) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_01 and SNP_07.

Thus, in one embodiment the plants according to the invention compriseat least a Adenine (A) (i.e. the AA or AT genotype) instead of twoThymines (TI) at nucleotide 71 of SEQ ID NO: 1 (referred to as SNP_01)or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO: 1; and/or at least aCytosine (C) (i.e. the CC or CT genotype) instead of two Thymines (TT)at nucleotide 71 of SEQ ID NO: 2 (referred to as SNP_02) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO:2; and/or at least a Adenine (A) (i.e.the AA or AC genotype) instead of two Cytosines (CC) at nucleotide 71 ofSEQ ID NO: 3 (referred to as SNP_03) or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO:3; and/or at least a Guanine (G) (i.e. the GG or GA genotype) insteadof two Adenines (AA) at nucleotide 71 of SEQ ID NO: 4 (referred to asSNP_04) or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:4; and/or at least a Thymine(T) (i.e. the TT or TC genotype) instead of two Cytosines (CC) atnucleotide 71 of SEQ ID NO: 5 (referred to as SNP_05) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO:5; and/or at least a Cytosine (C) (i.e.the CC or CA genotype) instead of two Adenines (AA) at nucleotide 71 ofSEQ ID NO: 6 (referred to as SNP_06) or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO:6; and/or at least a Guanine (G) (i.e. the GG or GT genotype) insteadof two Thymines (TI) at nucleotide 71 of SEQ ID NO: 7 (referred to asSNP_07) or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:7.

In another embodiment the presence of the introgression fragment, or thechromosome 6 region (or variant or orthologous chromosome 6 region),comprising QTL6.1 (or variant), is detectable by a molecular markerassay which detects at least 1, preferably at least 2, 3 or 4 of theSingle Nucleotide Polymorphism (SNP) markers selected from the groupconsisting of:

-   -   a) The CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP1.23 in SEQ ID NO: 23 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 23;    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 2;    -   c) the TT or CT genotype for the Single Nucleotide Polymorphism        marker SNP2.24 in SEQ ID NO: 24 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 24;    -   d) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 3;    -   e) any a L. virosa genome specific marker, in between marker        SNP1.23 and SNP_03.

Thus, in one embodiment the plants according to the invention compriseat least a Cytosine (C) (i.e. the CC or CT genotype) instead of twoThymines (TT) at nucleotide 71 of SEQ ID NO: 23 (referred to as SNP1.23)or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:23; and/or at least aCytosine (C) (i.e. the CC or CT genotype) instead of two Thymines (TI)at nucleotide 71 of SEQ ID NO: 2 (referred to as SNP_02) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO:2; and/or at least a Thymine (T) (i.e.the TT or CT genotype) instead of two Cytosines (CC) at nucleotide 71 ofSEQ ID NO: 24 (referred to as SNP2.24) or at the equivalent nucleotideof a genomic sequence comprising substantial sequence identity to SEQ IDNO:24; and/or at least a Adenine (A) (i.e. the AA or AC genotype)instead of two Cytosines (CC) at nucleotide 71 of SEQ ID NO: 3 (referredto as SNP_03) or at the equivalent nucleotide of a genomic sequencecomprising substantial sequence identity to SEQ ID NO:3.

In one embodiment the presence of the introgression fragment, or thechromosome 7 region (or variant or orthologous chromosome 7 region),comprising QTL7.2 (or variant), is detectable by a molecular markerassay which detects at least 1, preferably at least 2, 3, 4, 5, 6, ormore (or all 7) Single Nucleotide Polymorphism (SNP) markers selectedfrom the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:9);    -   c) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:10);    -   d) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 11);    -   e) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_12 in SEQ ID NO: 12 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 12);    -   f) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_13 in SEQ ID NO: 13 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:13);    -   g) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 14 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:14);    -   h) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_08 and SNP_14.

Thus, in one embodiment the plants according to the invention compriseat least a Thymine (T) (i.e. the TT or TC genotype) instead of twoCytosines (CC) at nucleotide 71 of SEQ ID NO: 8 (referred to as SNP_08)or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:8; and/or at least a Cytosine(C) (i.e. the CC or CT genotype) instead of two Thymines (TT) atnucleotide 71 of SEQ ID NO: 9 (referred to as SNP_09) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO:9; and/or at least a Adenine (A) (i.e.the AA or AG genotype) instead of two Guanines (GG) at nucleotide 71 ofSEQ ID NO: 10 (referred to as SNP_10) or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO:10; and/or at least a Cytosine (C) (i.e. the CC or CA genotype)instead of two Adenines (AA) at nucleotide 71 of SEQ ID NO: 11 (referredto as SNP_11) or at the equivalent nucleotide of a genomic sequencecomprising substantial sequence identity to SEQ ID NO: 11; and/or atleast a Cytosine (C) (i.e. the CC or CT genotype) instead of twoThymines (TT) at nucleotide 71 of SEQ ID NO: 12 (referred to as SNP_12)or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:12; and/or at least a Guanine(G) (i.e. the GG or GA genotype) instead of two Adenines (AA) atnucleotide 136 of SEQ ID NO: 13 (referred to as SNP_13) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO: 13; and/or at least a Cytosine (C) (i.e.the CC or CT genotype) instead of two Thymines (T) at nucleotide 71 ofSEQ ID NO: 14 (referred to as SNP_14) or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO: 14.

In one embodiment the presence of the introgression fragment, or thechromosome 7 region (or variant or orthologous chromosome 7 region),comprising QTL7.1 (or variant), is detectable by a molecular markerassay which detects at least 1, preferably at least 2, 3, 4, 5, 6, 7 ormore (or all 8) Single Nucleotide Polymorphism (SNP) markers selectedfrom the group consisting of:

-   -   a) the TT or TA genotype for the Single Nucleotide Polymorphism        marker SNP_15 in SEQ ID NO: 15 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:15);    -   b) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_16 in SEQ ID NO: 16 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:16);    -   c) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:17);    -   d) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:18);    -   e) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:19);    -   f) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:20);    -   g) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_21 in SEQ ID NO: 21 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:21);    -   h) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_22 in SEQ ID NO: 22 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:22);    -   i) any wild lettuce genome-specific marker, such as a L. virosa        genome specific marker, in between marker SNP_15 and SNP_22.

Thus, in one embodiment the plants according to the invention compriseat least a Thymine (T) (i.e. the TT or TA genotype) instead of twoAdenines (CC) at nucleotide 71 of SEQ ID NO: 15 (referred to as SNP_15)or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:15; and/or at least a Guanine(G) (i.e. the GG or GA genotype) instead of two Adenines (AA) atnucleotide 71 of SEQ ID NO: 16 (referred to as SNP_16) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO:16; and/or at least a Thymine (T) (i.e.the TT or TC genotype) instead of two Cytosines (CC) at nucleotide 71 ofSEQ ID NO: 17 (referred to as SNP_17) or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO: 17; and/or at least a Guanine (G) (i.e. the GG or GC genotype)instead of two Cytosines (CC) at nucleotide 71 of SEQ ID NO: 18(referred to as SNP_18) or at the equivalent nucleotide of a genomicsequence comprising substantial sequence identity to SEQ ID NO: 18;and/or at least a Guanine (G) (i.e. the GG or GA genotype) instead oftwo Adenines (AA) at nucleotide 71 of SEQ ID NO: 19 (referred to asSNP_19) or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:19; and/or at least a Thymine(T) (i.e. the TT or TC genotype) instead of two Cytosines (CC) atnucleotide 72 of SEQ ID NO: 20 (referred to as SNP_20) or at theequivalent nucleotide of a genomic sequence comprising substantialsequence identity to SEQ ID NO:20; and/or at least a Cytosine (C) (i.e.the CC or CA genotype) instead of two Adenines (AA) at nucleotide 41 ofSEQ ID NO: 21 (referred to as SNP_21) or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO:21; and/or at least a Cytosine (C) (i.e. the CC or CT genotype)instead of two Thymines (TT) at nucleotide 71 of SEQ ID NO: 22 (referredto as SNP_22) or at the equivalent nucleotide of a genomic sequencecomprising substantial sequence identity to SEQ ID NO:22.

In a further embodiment the presence of the introgression fragment, orthe chromosome 7 region (or variant or orthologous chromosome 7 region),comprising QTL7.1 (or variant), is detectable by a molecular markerassay which detects at least 1, preferably at least 2, 3, 4 of theSingle Nucleotide Polymorphism (SNP) markers selected from the groupconsisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 17);    -   b) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP17.25 in SEQ ID NO: 25 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 25);    -   c) the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18);    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19);    -   e) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_17 and SNP_19.

Thus, in one embodiment the plants according to the invention compriseat least a Thymine (T) (i.e. the TT or TC genotype) instead of twoCytosines (CC) at nucleotide 71 of SEQ ID NO: 17 (referred to as SNP_17)or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:17; at least a Thymine (T)(i.e. the TT or TC genotype) instead of two Cytosines (CC) at nucleotide71 of SEQ ID NO: 25 (referred to as SNP17.25) or at the equivalentnucleotide of a genomic sequence comprising substantial sequenceidentity to SEQ ID NO:25; and/or at least a Guanine (G) (i.e. the GG orGC genotype) instead of two Cytosines (CC) at nucleotide 71 of SEQ IDNO: 18 (referred to as SNP_18) or at the equivalent nucleotide of agenomic sequence comprising substantial sequence identity to SEQ IDNO:18; and/or at least a Guanine (G) (i.e. the GG or GA genotype)instead of two Adenines (AA) at nucleotide 71 of SEQ ID NO: 19 (referredto as SNP_19) or at the equivalent nucleotide of a genomic sequencecomprising substantial sequence identity to SEQ ID NO:19.

The SNP genotype refers to two nucleotides, and genomic sequencescomprising one of these two nucleotides, one on each chromosome 6 (forSNP_01 to SNP_07 or for SNP1.23, SNP_02, SNP2.24 or SNP_03) or 7 (forSNP_08 to SNP_14 and SNP_15 to SNP_22 or for SNP_17, SNP17.25, SNP_18 orSNP_19). So a plant having a CC genotype for SNP_22 has an identicalnucleotide (C) on both chromosomes, while a plant having an CT genotypefor SNP_22 has one chromosome with an C at nucleotide 71 of SEQ ID NO:22 (or at the equivalent nucleotide of a genomic sequence comprisingsubstantial sequence identity to SEQ ID NO:22) and one chromosome with aT at nucleotide 71 of SEQ ID NO: 22 (or at the equivalent nucleotide ofa genomic sequence comprising substantial sequence identity to SEQ IDNO:22). As the genomic sequences around the SNP markers provided hereinmay vary slightly in introgression fragments from other wild lettuceaccessions (i.e. variants or orthologous chromosome 6 or 7 regions) itis clear that the nucleotide sequences before and after the SNP may notbe 100% identical to the sequences provided herein. Therefore sequenceshaving substantial sequence identity to the sequences provided herein,but which comprise the same SNP, are encompassed herein. It is alsoclear that in certain aspects the introgression is in homozygous form,and the SNP marker genotype is then the genotype homozygous for the QTL.

The introgression fragment may be large, even half of a chromosome, orsmall, as long as the Nr: 1 resistance conferring part is retained. Inone aspect the introgression fragment on chromosome 6 and/or 7 is equalto or less than 120 Mb, 100 Mb, 84 Mb, 80 Mb, 75 Mb, 74 Mb, 73 Mb, 60Mb, 50 Mb, 40 Mb, 30 Mb, 20 Mb, 16 Mb, 15 Mb, 10 Mb in size, preferablyequal to or less than 8 Mb in size, more preferably equal to or lessthan 6, 5, 4, 3 or 2.5 Mb in size, e.g. equal to or less than 2 Mb. Inone aspect the introgression fragment is at least 0.2 Mb, 0.5 Mb, 1.0Mb, 1.5 Mb, 1.9 Mb, 2.0 Mb, 2.5 Mb or 3 Mb in size. Thus, various rangesof introgression sizes are encompassed herein. The size can be easilydetermined by e.g. whole genome sequencing or Next GenerationSequencing, e.g. as described in Qi et al. 2013 (Nature GeneticsDecember 2013, Vol 45, No. 12, pages 1510-1518) or in Huang et al. 2009(Nature Genetics, Volume 41, Number 12, p 1275-1283). Especiallyintrogression regions can be easily distinguished from cultivatedgenomic regions due to the larger amount of genetic variation (SNPs,INDELs, etc.) in the introgression region.

The skilled person knows how to screen and identify wild lettuce, e.g.L. virosa, for the presence of any one of the QTLs or orthologs orvariants as described herein. E.g. various L. virosa accessions canfirst be selected phenotypically by assaying their Nr:1 resistance,especially free-choice and/or non-choice resistance and in one aspectselect those accessions which comprise both free-choice and non-choiceresistance. Alternatively, various L. virosa accessions may be screeneddirectly for the presence of one or more of the SNP markers (or markersin between the SNP markers) described herein. Once a candidate wildlettuce, e.g. L. virosa, has been identified, the skilled person knowshow to transfer one, two or all three of the QTLs of the invention fromthe wild lettuce into cultivated lettuce using traditional breedingtechniques. For example, plants grown from the wild accessions, such asplants grown from deposited seeds (NCIMB42086), can be crossed with acultivated lettuce plant to obtain F1 seeds. The F1 plants can be selfedone or more times to produce F2 or F3 plants (or further selfinggenerations), and/or F1, F2 plants or F3 plants, etc., can bebackcrossed to a cultivated lettuce parent. Progeny plants whichcomprise the QTL6.1 (or variant) and/or QTL7.1 (or variant) and/orQTL7.2 (or variant) can be screened for, and selected for, by thepresence of one or more or all of the above SNP markers (or markers inbetween any of those markers) in order to identify plants comprising arecombinant chromosome 6 and/or 7, comprising the QTL(s). Techniquessuch as embryo rescue may need to used to obtain progeny frominterspecific crosses (e.g. between L. sativa and L. virosa).

In yet another embodiment of the invention the presence of theintrogression fragment in a cultivated lettuce plant, or the chromosome6 region (or orthologous chromosome 6 region), comprising QTL6.1 (orvariant), is detectable by a molecular marker assay which detects atleast one of the markers selected from the group consisting of:

-   -   a) the AA or AT genotype for the Single Nucleotide Polymorphism        marker SNP_01 in SEQ ID NO: 1 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 1);    -   b) the GG or GT genotype for the Single Nucleotide Polymorphism        marker SNP_07 in SEQ ID NO: 7 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:7);    -   c) any wild lettuce genome-specific marker in between marker        SNP_01 and SNP_07;    -   d) any wild lettuce genome-specific marker which is genetically        linked within 7 cM, 5 cM, 3 cM or less of any one of markers        SNP_01 to SNP_07; and    -   e) any wild lettuce genome-specific marker which is physically        linked within 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb or 0.2 Mb or less        of any one of markers SNP_01 to SNP_07.

In one aspect the markers of c) are one or more of SNP_02 to SNP_06.

In one aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b) and/or c) above. Inanother aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b), c), d) and/or e) above.In one embodiment at least the marker of a) and/or b) is detected andoptionally at least one, two, three or more markers of c), d) and/or e)are detected. In one aspect at least 1, 2, or 3 markers of c) aredetected, especially at least SNP_04, SNP_05 and/or SNP_06.

Any wild lettuce genome-specific marker (e.g. L. virosa genome specific)in-between the marker of a) and b) refers to any molecular marker whichmaps genetically to the chromosome 6 region in-between marker SNP_01 andSNP_07 and/or which lies physically in-between marker SNP_01 and SNP_07,and which is indicative of the wild lettuce chromosome 6 region. Thismeans that the marker is polymorphic between the cultivated lettucegenome and the wild lettuce genome. In one aspect, the marker is aSingle Nucleotide Polymorphism (SNP), but other molecular markers suchas RFLP, AFLP, RAPD, DNA sequencing, etc. may equally be used.

In an alternative embodiment of the invention the presence of theintrogression fragment in a cultivated lettuce plant, or the chromosome6 region (or orthologous chromosome 6 region), comprising QTL6.1 (orvariant), is detectable by a molecular marker assay which detects atleast one of the markers selected from the group consisting of:

-   -   a) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP1.23 in SEQ ID NO: 23 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:23);    -   b) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 3;    -   c) any L. virosa genome specific marker, physically located in        between marker SNP1.23 and SNP_03.    -   d) any L. virosa genome specific marker which is physically        linked within 10 Mb, 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb or 0.2 Mb or        less of any one of markers SNP1.23 to SNP_03.

In one aspect the markers of c) are one or more of SNP_02, SNP2.24,optionally VSP1 and/or VSP3.

In one aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b) and/or c) above. Inanother aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b), c), and/or d) above. Inone embodiment at least the marker of a) and/or b) is detected andoptionally at least one, two, three or more markers of c) and/or d) aredetected. In one aspect at least 1, 2, or 3 markers of c) are detected,especially at least SNP_02 and/or SNP2.24.

Any L. virosa genome specific marker means that the marker is indicativeof the introgression fragment and the presence of the L. virosa genome,i.e. the marker is polymorphic between the cultivated lettuce genome andthe wild L. virosa lettuce genome. In one aspect, the marker is a SingleNucleotide Polymorphism (SNP), but other molecular markers such as RFLP,AFLP, RAPD, DNA sequencing, etc. may equally be used.

Likewise in one embodiment of the invention the presence of theintrogression fragment in a cultivated lettuce plant, or the chromosome7 region (or orthologous chromosome 7 region), comprising QTL7.2 (orvariant), is detectable by a molecular marker assay which detects atleast one of the markers selected from the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 14 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:8);    -   c) any wild lettuce genome-specific marker in between marker        SNP_08 and SNP_14;    -   d) any wild lettuce genome-specific marker which is genetically        linked within 7 cM, 5 cM, 3 cM or less of any one of markers        SNP_08 to SNP_14; and    -   e) any wild lettuce genome-specific marker which is physically        linked within 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb or 0.2 Mb or less        of any one of markers SNP_08 to SNP_14.

In one aspect the markers of c) are one or more of SNP_09 to SNP_13.

In one aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b) and/or c) above. Inanother aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b), c), d) and/or e) above.In one embodiment at least the marker of a) and/or b) is detected andoptionally at least one, two, three or more markers of c), d) and/or e)are detected. In one aspect at least 1, 2 or 3 markers of c) aredetected, especially SNP_09, SNP_10 and/or SNP_11.

Any wild lettuce genome-specific marker (e.g. L. virosa genome specific)in-between the marker of a) and b) refers to any molecular marker whichmaps genetically to the chromosome 6 region in-between marker SNP_08 andSNP_14 and/or which lies physically in-between marker SNP_08 and SNP_14,and which is indicative of the wild lettuce chromosome 7 region. Thismeans that the marker is polymorphic between the cultivated lettucegenome and the wild lettuce genome. In one aspect, the marker is aSingle Nucleotide Polymorphism (SNP), but other molecular markers suchas RFLP, AFLP, RAPD, DNA sequencing, etc. may equally be used.

Likewise in one embodiment of the invention the presence of theintrogression fragment in a cultivated lettuce plant, or the chromosome7 region (or orthologous chromosome 7 region), comprising QTL7.1 (orvariant), is detectable by a molecular marker assay which detects atleast one of the markers selected from the group consisting of:

-   -   a) the TT or TA genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 15 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:15);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 22 (or in a sequence comprising        substantial sequence identity to SEQ ID NO:22);    -   c) any wild lettuce genome-specific marker in between marker        SNP_15 and SNP_22;    -   d) any wild lettuce genome-specific marker which is genetically        linked within 7 cM, 5 cM, 3 cM or less of any one of markers        SNP_15 to SNP_22; and    -   e) any wild lettuce genome-specific marker which is physically        linked within 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb or 0.2 Mb or less        of any one of markers SNP_15 to SNP_22.

In one aspect the markers of c) are one or more of SNP_16 to SNP_21.

In one aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b) and/or c) above. Inanother aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b), c), d) and/or e) above.In one embodiment at least the marker of a) and/or b) is detected andoptionally at least one, two, three or more markers of c), d) and/or e)are detected. In one aspect at least 1, 2 or 3 markers of c) aredetected, especially SNP_19, SNP_20 and/or SNP_21.

Any wild lettuce genome-specific marker (e.g. L. virosa genome specific)in-between the marker of a) and b) refers to any molecular marker whichmaps genetically to the chromosome 6 region in-between marker SNP_15 andSNP_22 and/or which lies physically in-between marker SNP_15 and SNP_22,and which is indicative of the wild lettuce chromosome 7 region. Thismeans that the marker is polymorphic between the cultivated lettucegenome and the wild lettuce genome. In one aspect, the marker is aSingle Nucleotide Polymorphism (SNP), but other molecular markers suchas RFLP, AFLP, RAPD, DNA sequencing, etc. may equally be used.

In an alternative embodiment of the invention the presence of theintrogression fragment in a cultivated lettuce plant, or the chromosome7 region (or orthologous chromosome 7 region), comprising QTL7.1 (orvariant), is detectable by a molecular marker assay which detects atleast one of the markers selected from the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 17);    -   b) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19);    -   c) any a L. virosa genome specific marker, physically located in        between marker SNP_17 and SNP_19.    -   d) any L. virosa genome specific marker which is physically        linked within 12 Mb, 10 Mb, 5 Mb, 3 Mb, 2 Mb, 1 Mb, 0.5 Mb or        0.2 Mb or less of any one of markers SNP_17 to SNP_19.

In one aspect the markers of c) are one or more of SNP17.25, SNP_18,optionally VSP4.

In one aspect the marker of d) is VSP2 or SNP_16.

In one aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b) and/or c) above. Inanother aspect, at least one, two, at least three, at least four or moremarkers are detected from the markers of a), b), c), and/or d) above. Inone embodiment at least the marker of a) and/or b) is detected andoptionally at least one, two, three or more markers of c) and/or d) aredetected. In one aspect at least 1, 2, or 3 markers of c) are detected,especially at least SNP17.25 and/or SNP_18.

Any L. virosa genome specific marker means that the marker is indicativeof the introgression fragment and the presence of the L. virosa genome,i.e. the marker is polymorphic between the cultivated lettuce genome andthe wild L. virosa lettuce genome. In one aspect, the marker is a SingleNucleotide Polymorphism (SNP), but other molecular markers such as RFLP,AFLP, RAPD, DNA sequencing, etc. may equally be used.

Also provided are seeds from which a plant of the invention can begrown, as are lettuce leaves (or parts thereof) and heads harvested froma plant of the invention and comprising the recombinant chromosome 6and/or 7 in their genome. Likewise a plant cell, tissue or plant part ofa plant or of a seed is provided comprising at least one recombinantchromosome 6 and/or 7, wherein said recombinant chromosome 6 and/or 7comprises an introgression fragment from a wild lettuce and wherein saidintrogression fragment comprises a QTL conferring Nr:1 resistance.

The molecular markers described herein may be detected according tostandard method. For example SNP markers can easily be detected using aKASP-assay (see www.kpbioscience.co.uk) or other assays. For developinga KASP-assay, for example 70 base pairs upstream and 70 base pairsdownstream of the SNP can be selected and two allele-specific forwardprimers and one allele specific reverse primer can be designed. See e.g.Allen et al. 2011, Plant Biotechnology J. 9, 1086-1099, especially p097-1098 for KASP assay method.

Thus, in one aspect, the SNP markers and the presence/absence of themarker associated with the QTL(s) is determined using a KASP assay, butequally other assays can be used. For example, optionally DNA sequencingmay also be used.

The physical size of an introgression fragment can be determined byvarious methods, such as physical mapping, sequencing or byvisualization of the introgression using Fluorescent in situhybridization (FISH) images (Verlaan et al. 2011, Plant Journal 68:1093-1103).

Plants with various sizes introgression fragments on chromosome 6 and/or7 can be generated by generating recombinant plants from a population ofplants derived from a cross between a cultivated lettuce plant (lackingthe introgressions) and a Nr:1 resistant L. virosa plant or betweencultivated lettuce and a plant of the invention (a cultivated lettucecomprising a recombinant chromosome 6 and/or 7) and selecting progenyhaving different introgression sizes.

Methods

The markers and genomic regions identified herein can be used in variousmethods, and this applies to the first regions and markers identified(see e.g. FIG. 3A) as well as to the later regions and markersidentified (see e.g. FIG. 3B), because the QTLs are the same. Theinvention provides a number of methods, namely:

1) a method for identifying wild lettuce plant, especially a L. virosaaccession, comprising one or more of QTL6.1, QTL7.1 and/or QTL7.2 (orvariants of any of these);

2) a method for transferring one or more of the QTLs selected fromQTL6.1, QTL7.1 and/or QTL7.2 (or variants of any of these) from a wildlettuce plant (e.g. L. virosa) into cultivated lettuce (L. sativa) togenerate a Nr: 1 resistant cultivated lettuce;

3) a method for screening cultivated lettuce lines or varieties for thepresence of one or more of the QTLs selected from QTL6.1, QTL7.1 and/orQTL7.2 (or variants of any of these); and

4) a method for transferring one or more of the QTLs selected fromQTL6.1, QTL7.1 and/or QTL7.2 (or variants of any of these) from acultivated lettuce (L. sativa) into another cultivated lettuce, e.g.into a Nr: 1 susceptible lettuce plant line or variety;5) a method for using seeds deposited under accession number NCIMB42086,or descendants thereof, for generating Nr:1 resistant cultivatedlettuce;6) a method for cultivating plants of the invention, i.e. Nr:1 resistantL. sativa plants comprising one or more of the QTLs selected fromQTL6.1, QTL7.1 and/or QTL7.2 (or variants of any of these), in areaswhere N. ribisnigri biotype Nr: 1 is present.Method for Identifying Wild Lettuce Comprising One or More of QTL6.1.QTL7.1 and/or QTL7.2 (or Variants of any of these)

In one aspect a method for identifying wild lettuce plants comprisingone or more of QTL6.1, QTL7.1 and/or QTL7.2 (or variants thereof) isprovided, comprising the steps of:

-   -   a) providing a wild lettuce plant or a plurality of wild lettuce        plants;    -   b) optionally testing the wild lettuce plant or plurality of        plants for Nr:1 resistance in an Nr:1 resistance assay;    -   c) screening the genomic DNA of the plant or plurality of plants        of a), or optionally only of the Nr:1 resistant plant or plants        identified in b), for the presence of one or more markers        indicative of QTL6.1 or a variant thereof and/or indicative of        QTL7.1 or a variant thereof and/or indicative of QTL7.2 or a        variant thereof; and    -   d) identifying a plant comprising one or more of said markers of        c);    -   e) optionally testing the plant of d) for Nr:1 resistance in an        Nr: 1 resistance assay;    -   f) optionally crossing the wild lettuce plant of d) with a        cultivated lettuce plant.

Optionally the method further comprises introgressing the QTL6.1, QTL7.1and/or QTL7.2 (or variants thereof) into cultivated lettuce, especiallyNr:1 susceptible lettuce, and generating a L. sativa plant comprisingNr:1 resistance conferred by one or more of the introgression fragments.This can e.g. be done by backcrossing. Optionally marker assistedselection may be used.

The plant or plants in step a) are preferably L. virosa, e.g.originating from different geographic regions.

In step b) or e) a phenotypic Nr: resistance assay (e.g. field test orcontrolled environment test) can be carried out in order to selectplants which are resistant against Nr:1 and therefore putativelycomprise one or more of the QTLs. The Nr: 1 resistance assay may be afree choice and/or non-choice assay.

A Nr: 1 resistant L. sativa plant obtained by the method is also anembodiment of the invention.

The genomic DNA in step c) can be screened for the presence of one ormore markers indicative of QTL6.1 or a variant thereof, as describedfurther above, e.g. by determining the presence of one or more markersselected from the group consisting of:

-   -   a) the AA or AT genotype for the Single Nucleotide Polymorphism        marker SNP_01 in SEQ ID NO: 1 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 1; and/or    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 2; and/or    -   c) the AA or AC genotype for the Single Nucleotide Polymorphism        marker SNP_03 in SEQ ID NO: 3 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 3; and/or    -   d) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_04 in SEQ ID NO: 4 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 4; and/or    -   e) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_05 in SEQ ID NO: 5 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 5; and/or    -   f) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_06 in SEQ ID NO: 6 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 6; and/or    -   g) the GG or GT genotype for the Single Nucleotide Polymorphism        marker SNP_07 in SEQ ID NO: 7 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 7; and/or    -   h) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_01 and SNP_07, e.g. in between any two markers of SNP_01 to        SNP_07.

Alternatively, the genomic DNA in step c) can be screened for thepresence of one or more markers indicative of QTL6.1 or a variantthereof, as described further above, e.g. by determining the presence ofone or more markers selected from the group consisting of:

-   -   a) The CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP1.23 in SEQ ID NO: 23 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 23);    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_02 in SEQ ID NO: 2 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 2);    -   c) the TT or CT genotype for the Single Nucleotide Polymorphism        marker SNP2.24 in SEQ ID NO: 24 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 24);        -   the AA or AC genotype for the Single Nucleotide Polymorphism            marker SNP_03 in SEQ ID NO: 3 (or in a sequence comprising            substantial sequence identity to SEQ ID NO: 3);    -   d) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP1.23 and SNP_03 (e.g. in-between SNP1.23 and SNP2.24, SNP1.23        and SNP_02); or in between SNP_02 and SNP_03 (e.g. in-between        SNP_02 and SNP2.24); or in between SNP2.24 and SNP_03;    -   e) any wild lettuce genome specific marker especially L.        virosa-genome specific marker, located within a distance of 10        Mb, preferably within 5 Mb, of any marker selected from SNP1.23,        SNP_02, SNP2.24, or SNP_03.

Optionally also L. virosa specific markers (VSP1 or VSP3) can bescreened to identify and/or distinguish accession types comprising theQTL.

The genomic DNA in step c) can be screened for the presence of one ormore markers indicative of QTL7.2 or a variant thereof, as describedfurther above, e.g. by determining the presence of one or more markersselected from the group consisting of:

-   -   a) the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_08 in SEQ ID NO: 8 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 8;    -   b) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_09 in SEQ ID NO: 9 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 9;    -   c) the AA or AG genotype for the Single Nucleotide Polymorphism        marker SNP_10 in SEQ ID NO: 10 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 10;    -   d) the CC or CA genotype for the Single Nucleotide Polymorphism        marker SNP_11 in SEQ ID NO: 11 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 11;    -   e) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_12 in SEQ ID NO: 12 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 12;    -   f) the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_13 in SEQ ID NO: 13 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 13;    -   g) the CC or CT genotype for the Single Nucleotide Polymorphism        marker SNP_14 in SEQ ID NO: 14 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 14;    -   h) any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_08 and SNP_14, e.g. in between any two markers of SNP_08 to        SNP_14.

The genomic DNA in step c) can be screened for the presence of one ormore markers indicative of QTL7.1 or a variant thereof, as describedfurther above, e.g. by determining the presence of one or more markersselected from the group consisting of:

-   -   i. the TT or TA genotype for the Single Nucleotide Polymorphism        marker SNP_15 in SEQ ID NO: 15 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 15;    -   ii. the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_16 in SEQ ID NO: 16 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 16;    -   iii. the TT or TC genotype for the Single Nucleotide        Polymorphism marker SNP_17 in SEQ ID NO: 17 or in a sequence        comprising substantial sequence identity to SEQ ID NO: 17;    -   iv. the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18;    -   v. the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19;    -   vi. the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_20 in SEQ ID NO: 20 or in a sequence comprising        substantial sequence identity to SEQ ID NO: 20;    -   vii. the CC or CA genotype for the Single Nucleotide        Polymorphism marker SNP_21 in SEQ ID NO: 21 or in a sequence        comprising substantial sequence identity to SEQ ID NO: 21;    -   viii. the CC or CT genotype for the Single Nucleotide        Polymorphism marker SNP_22 in SEQ ID NO: 22 or in a sequence        comprising substantial sequence identity to SEQ ID NO: 22;    -   ix. any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_15 and SNP_22, e.g. in between any two markers of SNP_15 to        SNP_22.

Alternatively, the genomic DNA in step c) can be screened for thepresence of one or more markers indicative of QTL7.1 or a variantthereof, as described further above, e.g. by determining the presence ofone or more markers selected from the group consisting of:

-   -   the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP_17 in SEQ ID NO: 17 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 17);    -   the TT or TC genotype for the Single Nucleotide Polymorphism        marker SNP17.25 in SEQ ID NO: 25 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 25);    -   the GG or GC genotype for the Single Nucleotide Polymorphism        marker SNP_18 in SEQ ID NO: 18 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 18);    -   the GG or GA genotype for the Single Nucleotide Polymorphism        marker SNP_19 in SEQ ID NO: 19 (or in a sequence comprising        substantial sequence identity to SEQ ID NO: 19);    -   any wild lettuce genome specific marker, especially L.        virosa-genome specific marker, located physically in-between        SNP_17 and SNP_19 (e.g. in-between SNP_17 and SNP_18, in between        SNP_17 and SNP17.25; or in between SNP17.25 and SNP_19, or in        between SNP17.25 and SNP_18, or in between SNP_18 and SNP_19);    -   any wild lettuce genome specific marker especially L.        virosa-genome specific marker, located within a distance of 12        Mb, 10 Mb, preferably within 5 Mb, of any marker selected from        SNP_17, SNP_17.25, SNP_18 and SNP_19.

Optionally also L. virosa accession specific markers can be screened(VSP2 or VSP4) to distinguish accession types comprising the QTL.

The marker screening can be done by any suitable technique orcombination of techniques known to the skilled person, e.g. PCR-based,sequencing based, etc. It is understood that screening of the genomicDNA can be done on plants, plant parts, seeds or on genomic DNA isolatedtherefrom.

In step d) of the method a plant is identified comprising one or more ofthe markers, e.g. at least 1, 2, 3, 4, 5, 6 or all 7 of SNP_01 to SNP_07and/or any marker in-between SNP_01 and SNP_07; or alternatively one ormore of the markers of SNP1.23, SNP_02, SNP2.24 or SNP_03 (andoptionally VSP1 or VSP3) and/or any marker in-between SNP1.23 andSNP_03; at least at least 1, 2, 3, 4, 5, 6 or all 7 of SNP_08 to SNP_14and/or any marker in-between SNP_08 and SNP_14; at least 1, 2, 3, 4, 5,6, 7 or all 8 of SNP_15 to SNP_22 and/or any marker in-between SNP_15and SNP_22; or alternatively SNP_17, SNP17.25, SNP_18 or SNP_19 (andoptionally VSP2 or VSP4) or any marker in between SNP_17 and SNP_19.

Thus, in one aspect a method for generating a cultivated lettuce plantcomprising Nr:1 resistance is provided comprising the steps of:

-   -   a) Providing a wild lettuce, especially a Lactuca virosa plant        comprising 1, 2, 3, 4, 5, or more SNP markers indicative of        QTL6.1 (or variant); and/or 1, 2, 3, 4, 5, or more SNP markers        indicative of QTL7.2 (or variant) and/or 1, 2, 3, 4, 5, or more        SNP markers indicative of QTL7.1 (or variant);    -   b) Crossing said wild lettuce, especially said Lactuca virosa        plant with a cultivated lettuce plant, which is susceptible        against lettuce aphid Nr: 1, to produce F1 seeds;    -   c) Optionally selfing the plants grown from F1 seeds one or more        times to produce F2, F3 or further generation selfing progeny;    -   d) Crossing said F1 or further generation selfing progeny to the        cultivated lettuce plant of step b), to produce a backcross        progeny;    -   e) Selecting backcross progeny which comprise resistance against        biotype Nr: 1.

A lettuce plant produced by the method is also encompassed herein.

Method for Transferring One or More of the QTLs Selected from QTL6.1.QTL7.1 and/or QTL7.2 (or Variants Thereof) from a Wild Lettuce (e.g. L.virosa) into Cultivated Lettuce (L. sativa) to Generate a Nr:1 ResistantCultivated Lettuce

In another aspect a method for transferring one or more of the QTLsselected from QTL6.1, QTL7.1 and/or QTL7.2 (or variants thereof) from awild lettuce (e.g. L. virosa) into cultivated lettuce (L. sativa) togenerate a Nr:1 resistant cultivated lettuce is provided, comprising:

-   -   a) providing a wild lettuce plant comprising QTL6.1, QTL7.1        and/or QTL7.2 (or variants thereof);    -   b) crossing the wild lettuce plant of a) with a cultivated        lettuce plant to generate an F1;    -   c) optionally selfing the F1 one or more times to generate        further selfing progeny;    -   d) backcrossing the F1 or further selfing progeny one or more        times to the cultivated lettuce plant of step b) (the recurrent        parent);    -   e) identifying and/or selecting backcross progeny comprising a        genome of the cultivated lettuce plant of step b) (the recurrent        parent) comprising an introgression fragment from the wild        lettuce plant of a) (donor parent) on chromosome 6 and/or on        chromosome 7.

In one aspect the wild lettuce plant of a) is a L. virosa. In one aspectthe L. virosa is Nr:1 resistant when tested in a Nr:1 resistance assay.In one aspect the L. virosa parent of a) is NCIMB42086, or progenythereof obtained by selfing and/or crossing, wherein the progenycomprise QTL6.1, QTL7.1 and/or QTL7.2. In another aspect the plant of a)is a L. virosa accession comprising the virosa specific markers VSP1and/or VSP2 or comprising the virosa specific markers VSP3 and/or VSP4(as shown in Table 6 and 7).

The cultivated lettuce of step b) in the method above (and in any othermethod of the invention) may be any L. sativa, such as an inbred line ora variety. It may be of any type, such as leaf or looseleaf, butterheador Bibb, Romaine or Cos, Crisphead or Iceberg, Celtuce or Stem lettuce.It is preferably a Nr: 1 susceptible plant, although it may also be aplant comprising Nr: 1 resistance conferred by different loci, in orderto stack Nr:1 resistance loci in one plant line or variety. It may be anNr:0 resistant plant. It may comprise the dominant Nr-gene.

When referring to backcrossing and backcross progeny, this may alsoinclude progeny obtained by backcrossing (BC) and selfing (S), e.g.BC1S1, BC1S2, BC2S1, etc.

In step e) any of the markers and marker assays described herein can beused.

Plants obtained by the method are also an embodiment of the invention,as described elsewhere herein. These plants are, thus, cultivated L.sativa plants (of any type) comprising one or more of the QTLs of theinvention on chromosome 6 (QTL6.1 or variant) and/or 7 (QTL7.1 and/orQTL7.2 or variants) in homozygous or heterozygous form.

As sterility barriers may exist between L. sativa and wild lettuceplants, such as L. virosa, the L. virosa plant (e.g. plants grown fromseeds having accession number NCIMB 42086 or other L. virosa accessions)may be crossed with a bridge species, such as L. serriola (Eenink et al.1982, Euphytica 31:291-299), and/or other methods, such astetraploidization, may be used to overcome sterility barriers. Thompsonand Ryder (1961; US Department of Agriculture, Tech. Bulletin no. 1244),for example, crossed L. virosa with a (L. serriola×L. sativa) hybrid,which produced a sterile F1 interspecific hybrid. However,tetraploidisation of the F1 and subsequent crossing and diploidizationenabled to introgress traits from L. virosa into L. sativa. Also embryorescue may be used to recover viable embryos from interspecific crosses(Maisonneuve et al. 1995, Euphytica 85: 281-285). Thus, when referringanywhere in the specification to a cultivated lettuce plants (L. sativa)comprising one or more QTLs conferring Nr:1 resistance obtainable bycrossing an L. virosa plant with a cultivated lettuce plant, this maycomprise (but is not limited to) steps which overcome sterilitybarriers, such as the use of a bridge species, embryo rescue and/orcolchicine treatment (chromosome doubling).

Method for Screening Cultivated Lettuce Lines or Varieties for thePresence of One or More of the QTLs Selected from QTL6.1, QTL7.1 and/orQTL7.2 (or Variants of any of these)

This method is similar to the method for identifying a wild lettuceplant comprising one or more of the QTLs, but herein cultivated lettuceplants, i.e. L. sativa plants, are screened.

The method thus comprises the following steps:

-   -   a) providing a cultivated lettuce plant or a plurality of        cultivated lettuce plants;    -   b) optionally testing the cultivated lettuce plant or plurality        of plants for Nr:1 resistance in an Nr: 1 resistance assay (e.g.        free-choice and/or non-choice);    -   c) screening the genomic DNA of the plant or plurality of plants        of a), or optionally only of the Nr:1 resistant plant or plants        identified in b), for the presence of one or more markers        indicative of QTL6.1 or a variant thereof and/or indicative of        QTL7.1 or a variant thereof and/or indicative of QTL7.2 or a        variant thereof; and    -   d) identifying a plant comprising one or more of said markers of        c);    -   e) optionally testing the plant of d) for Nr:1 resistance in an        Nr: 1 resistance assay.

Using this method for example commercial competitor varieties can bescreened, in order to determine whether they contain one or more of theQTLs of the instant invention.

Method for Transferring One or More of the QTLs Selected from QTL6.1,QTL7.1 and/or QTL7.2 (or Variants Thereof) from a Cultivated Lettuce (L.sativa) into Another Cultivated Lettuce, e.g. into a Nr:1 SusceptibleLettuce Plant Line or Variety

The QTLs of the present invention can off course also be transferredfrom one cultivated L. sativa plant to another cultivated L. sativaplant, in order to generate different types and different varieties oflettuce which are Nr: 1 resistant.

This method comprises the steps of:

-   -   a) providing a L. sativa plant comprising one or more or all of        QTL6.1, QTL7.1 and/or QTL7.2 or a variant of any of these;    -   b) crossing the L. sativa plant of a) with a second L. sativa        plant;    -   c) collecting F1 seeds from said cross and optionally selfing        said F1 plants one or more times to produce an F2 or F3 or        further selfing population,    -   d) optionally backcrossing the F1 plant or an F2 or F3 or        further selfing plant to the second L. sativa plant of b) to        produce a backcross population,    -   e) optionally selfing the backcross population one or more        times,    -   f) identifying a F1, F2, F3, further selfing or backcross plant        which comprises one or more or all of the SNP marker genotype        indicative of the introgression fragment on chromosome 6        (QTL6.1) and/or indicative of the introgression fragment on        chromosome 7 (QTL7.1 and/or QTL7.2).

Introgression fragments comprising QTL6.1, QTL7.1 and QTL7.2 (orvariants of any of these) can be transferred all together orindividually into another cultivated lettuce plant.

In one aspect the second cultivated lettuce plant of b) is a Nr: 1susceptible lettuce plant, or at least a plant lacking the QTL(s) whichit is to receive from the donor of a).

The new lettuce plant produced may again be any type and any line orvariety. Thus, the QTL(s) may be transferred by traditional breedingfrom one cultivated lettuce to another, e.g. from butterhead to romaine,from a stem lettuce to a Bibb, from a Romaine to a looseleaf, etc. Inthe course of the transfer the size of the introgression fragment may bereduced by recombination, and some of the markers may thereby not bepresent in the resulting plant.

Plants produced by this method are also an embodiment of the invention.

Thus, a method is provided for generating a cultivated lettuce plantcomprising Nr:1 resistance comprises the steps of:

-   -   a) Providing a cultivated lettuce plant comprising 1, 2, 3, 4,        5, or more SNP markers indicative of QTL6.1 (or a variant);        and/or 1, 2, 3, 4, 5, or more SNP markers indicative of QTL7.2        (or a variant) and/or 1, 2, 3, 4, 5, or more SNP markers        indicative of QTL7.1 (or a variant);    -   b) Crossing said cultivated lettuce plant with another        cultivated lettuce plant, which is susceptible against lettuce        aphid Nr:1 to produce F1 seeds;    -   c) Optionally selfing the plants grown from F1 seeds one or more        times to produce F2, F3 or further generation selfing progeny;    -   d) Identifying lettuce plants grown from F1, F2, F3 or further        generation selfing progeny which have a Nr: 1 resistance        phenotype and/or which comprise the introgression fragment or a        resistance-conferring part of the introgression fragment;    -   e) Optionally crossing said identified F1 progeny or selfing        progeny to the cultivated lettuce plant of step b), to produce a        backcross progeny;    -   f) Optionally selecting backcross progeny which comprises        resistance against biotype Nr:1 and/or which comprise the        introgression fragment or a resistance-conferring part of the        introgression fragment.

In step d) and/or f) markers described herein can be used.

A lettuce plant produced by the method is also encompassed herein.

Method for Using Seeds Deposited Under Accession Number NCIMB42086. OrDescendants Thereof. For Generating Nr:1 Resistant Cultivated Lettuce

NCIMB42086 comprises all three QTLs in homozygous form and can thus beused to generate cultivated lettuce lines or varieties comprising one ormore of the QTLs, as already described. Likewise descendants ofNCIMB42086 which retain one or more of the QTLs can be used to generatecultivated lettuce lines or varieties comprising one or more of theQTLs.

Method for Cultivating Plants of the Invention. i.e. Nr:1 Resistant L.sativa Plants Comprising One or More of the QTLs Selected from QTL6.1,QTL7.1 and/or QTL7.2 (or Variants Thereof). In Areas where N. RibisnigriBiotype Nr: 1 is Present

The plants of the invention can be cultivated in areas of natural Nr:1infestation. As the QTLs identified herein provide resistance against N.ribisnigri biotype Nr:1 not only under free-choice conditions, but alsoas under non-choice conditions, the resistance is very effective in thefield, because one can expect good yields even in situations where theinsects have no alternative choice for feeding and reproduction.Resistance which is present under only free-choice conditions is riskyto use, as aphids may still choose the plants for feeding andreproduction in situations where no other preferred choice is available.

Further, the QTLs of the invention were shown to provide resistanceagainst different isolates of biotype Nr:1, originating from differentcountries, such as Germany, France and Spain. The resistance istherefore expected to be effective and durable in Germany, France,Spain, UK and other European countries, as well as other countries ofthe world where biotype Nr: 1 may be found.

In one aspect the QTLs of the invention also provide resistance againstbiotype Nr:0, at least against European biotypes Nr:0 (i.e. at leastagainst German, French and Spanish biotypes Nr:1). Thus, in one aspectthe cultivated lettuce plants of the invention (comprising one or moreof the QTLs) are resistant against Nr:1 and at least also againstEuropean biotypes of Nr:0. In one aspect the plants are susceptibleagainst US or Californian biotypes of Nr:0, although this has yet to betested.

The field resistance of plants of the invention (comprising one or moreof the QTLs) against biotype Nr: 1, is significantly higher (i.e. asmeasurable by significantly lower average numbers of aphids) than forsusceptible controls, such as Mafalda. This can, for example, be testedin open-field tests in areas of natural Nr:1 infestation (e.g. inMurcia, Spain) by seeding or planting lettuce plants of the line orvariety comprising one or more of the introgression fragments in theirgenome in the field, together with suitable controls, such assusceptible variety Mafalda and/or a genetic control line. Preferably atleast about 10, 15, 20 or more plants per line or variety are included,as well as at least two or preferably three replicates. The plants canbe monitored weekly and once sufficient infestation is seen on thesusceptible control (e.g. at least 100 or more aphids), the numbers oflettuce aphids on a representative number of plants of each line orvariety can be counted. Preferably, in field conditions, the averagenumber of aphids of biotype Nr: 1 is significantly lower on plants ofthe invention compared to susceptible controls, such as Mafalda. Theaverage number of aphids is preferably not determined on young seedlings(below the 3-4 true leaf stage), as it was found in the Examples that onsuch young plants the resistance is not fully expressed yet.

Although plants of NCIMB42086 (comprising all three QTLs in homozygousform) were found to be completely free of Nr:1 aphids in free-choice andnon-choice field tests carried out in Spain, it may be (without beinglimited by speculation) that a cultivated lettuce plant comprising onlyone or two of the QTLs (or variants), or not all three introgressionscomprising the QTLs (or variants) in homozygous form, may not becompletely resistant against Nr: 1. Therefore, in one aspect, cultivatedlettuce plants of the invention, comprising one or more of the QTLs(QTL6.1, QTL7.1 and/or QTL7.2 or variants of any of these) in homozygousor heterozygous form, comprise an average number of aphids of biotypeNr: 1 is equal to or less than 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%40%, 30%, 20%, 10%, 5%, 3%, 2% or 1%, of the average number of aphidsfound on variety Mafalda (or a different Nr:1 susceptible variety,preferably comprising the Nr gene), or on the genetic control, whengrown under the same conditions. For example a free choice or anon-choice trial can be done in the field as described in the Examplesin order to determine the average number of Nr: 1 aphids on plants ofthe invention and on the control plants.

In one aspect the cultivated lettuce plants of the invention compriseequal to or less than an average of 50 N. ribisnigri biotype Nr: 1aphids, or equal to or less than an average of 40, 30, 20, or 10 Nr:1aphids. In another aspect the cultivated lettuce plants of the inventioncomprise (on average) zero Nr: 1 aphids or essentially zero Nr:1 aphids(equal to or less than 5 aphids on average) when grown in the field,while the control plant, such as Mafalda, comprises significant numbersof biotype Nr: 1 aphids. A significant number is at least 100 aphids onaverage, 150, 200, 250 or more.

In another aspect the cultivated lettuce plants of the inventioncomprise equal to or less than an average of 50 N. ribisnigri aphids (ofany biotype, i.e. biotype Nr:0 and biotype Nr:1), or equal to or lessthan an average of 40, 30, 20, or 10 aphids of N. ribisnigri aphids. Inanother aspect the cultivated lettuce plants of the invention comprise(on average) zero aphids N. ribisnigri aphids or essentially zero aphids(equal to or less than 5 aphids on average) when grown in the field inan area where both Nr: 1 and Nr:0 are present, while the control plant,such as Mafalda, comprises significant numbers of biotype Nr: 1 aphidsand while a Nr:0 susceptible variety comprises significant numbers ofbiotype Nr:0. A significant number is at least 100 aphids on average,150, 200, 250 or more.

Cultivated lettuce plants of the invention may be of any type. They maybe green lettuce or red lettuce, green and red lettuce (e.g. spotted),babyleaf, little-gem type lettuce, loose-leaf lettuce (also referred toas cutting or bunching lettuce), butterhead lettuce, Bibb lettuce,Batavia (or Summercrisp) lettuce, heading lettuce, romaine (or cos)lettuce, crisphead (or iceberg) lettuce, multileaf lettuce, Great LakesGroup lettuce, Vanguard Group lettuce, Salinas Group lettuce, Eastern(Ithaca) Group lettuce, Celtuce or Stem or Latin lettuce types, etc.They may also be of inter-market type, e.g. a cos with iceberg featuresfeatures, or a iceberg with cos features, etc. They may be inbred lines,F1 hybrids, double haploids, transgenic plants, mutant plants, etc.

In one aspect the introgression fragment(s) comprising one or more ofQTLs 6.1, 7.1 and/or 7.2 (or variants) is in homozygous form in thecultivated lettuce plant of the invention. Selfing one or more timeswill ensure that the introgression fragments are in homozygous form andthe SNP marker(s) then also show the homozygous genotype.

In a further aspect the cultivated lettuce plant of the invention hasgood fertility and is easily crossable with other cultivated lettucelines or varieties. Preferably any wild genome fragments (e.g. L.virosa) which are co-introduced with the QTL(s) and which confer anynegative characteristics in the cultivated plant, such as low fertilityand/or dwarf growth, are removed. This can be done by selectingrecombinants having a shorter introgression fragment, but which retainthe Nr:1-resistance conferring part.

Plants of the invention can be used to generate progeny (ordescendants), which have or retain the QTL(s) (or variants) and the Nr:1resistance phenotype. To generate progeny, a cultivated lettuceaccording to the invention can be selfed and/or crossed one or moretimes with another lettuce plant and seeds can be collected.

Also seeds from which any of the plants of the invention can be grownare provided.

In one embodiment, the use of a lettuce plant, of which representativeseeds have been deposited under accession number NCIMB 42086, or progenythereof (e.g. obtained by selfing), for generating a Nr: 1 resistantcultivated lettuce plant is provided.

In another embodiment, the use of cultivated lettuce plant comprising aNr:1 resistance phenotype conferred by one or more QTL(s) as found in/asobtainable from seeds deposited under accession number NCIMB 42086, orfrom progeny thereof (e.g. obtained by selfing), for generating a Nr:1resistant cultivated lettuce plant is provided.

Seed

Also seeds from which any of the plants of the invention can be grownare provided, as are containers or packages containing or comprisingsuch seeds. Seeds can be distinguished from other seeds due to thepresence of the one or more QTLs (as can be tested using molecularmarker tests described herein) and phenotypically.

In one aspect, seeds are packaged into small and/or large containers(e.g., bags, cartons, cans, etc.). The seeds may be pelleted prior topacking (to form pills or pellets) and/or treated with variouscompounds, such as seed coatings.

Seed pelleting can be combined with film coating (Halmer, P. 2000.Commercial seed treatment technology. In: Seed technology and itsbiological basis. Eds: Black, M. and Bewley, J. D., pages 257-286).Pelleting creates round or rounded shapes, which are easily sown withmodern sowing machines. A pelleting mixture typically contains seeds andat least glue and filler material. The latter could be, for example,clay, mica, chalk or cellulose. In addition, certain additives can beincluded to improve particular properties of the pellet, e.g., a seedtreatment formulation comprising at least one insecticidal, acaricidal,nematicidal or fungicidal compound can be added directly into thepelleting mixture or in separate layers. A seed treatment formulationcan include one of these types of compounds only, a mixture of two ormore of the same type of compounds or a mixture of one or more of thesame type of compounds with at least one other insecticide, acaricide,nematicide or fungicide.

Formulations especially suitable for the application as a seed treatmentcan be added to the seed in the form of a film coating including alsothe possibility of using the coating in or on a pellet, as well asincluding the seed treatment formulation directly into the pelletmixture. Characteristically, a film coating is a uniform, dust-free,water permeable film, evenly covering the surface of all individualseeds (Halmer, P. 2000. Commercial seed treatment technology. In: Seedtechnology and its biological basis. Eds: Black, M. and Bewley, J. D.,pages 257-286). Besides the formulation, the coating mixture generallyalso contains other ingredients such as water, glue (typically apolymer), filler materials, pigments and certain additives to improveparticular properties of the coating. Several coatings can be combinedon a single seed.

In addition, several combinations with film coating are possible: thefilm coating can be added on the outside of the pellet, in between twolayers of pelleting material, and directly on the seed before thepelleting material is added. Also more than 1 film coating layer can beincorporated in a single pellet. A special type of pelleting isencrusting. This technique uses less filler material, and the result isa ‘mini-pellet’.

Seeds may also be primed. Of all the commercially planted vegetableseeds, lettuce is most often primed. Priming is a water-based processthat is performed on seeds to increase uniformity of germination andemergence from the soil, and thus enhance vegetable stand establishment.Priming decreases the time span between the emergence of the first andthe last seedlings. Methods how to prime lettuce seeds are well known inthe art (see, e.g., Hill et al HortScience 42(6): 1436, 2007).

Plant parts and Vegetative Reproductions

In a further aspect plant parts, obtained from (obtainable from) a plantof the invention are provided herein, and containers or packagescomprising said plant parts. Any plant parts can be distinguished fromother lettuce plant parts by the presence of a recombinant chromosome 6and/or 7, i.e. by the presence of an introgression fragment from a wildlettuce, e.g. from L. virosa, on chromosome 6 and/or 7. This can beeasily tested by the presence of one or more or all of the markersdescribed herein.

In a preferred embodiment the plant parts are leaves or heads ofcultivated lettuce plants of the invention, preferably harvested leavesor heads, or parts of these.

Other plant parts, of plants of the invention, include stems, cuttings,petioles, cotyledons, flowers, anthers, pollen, ovaries, roots, roottips, protoplasts, callus, microspores, stalks, ovules, shoots, seeds,embryos, embryo sacs, cells, meristems, buds etc. Seeds include forexample seeds produced on the plant of the invention afterself-pollination or after pollination with pollen from another lettuceplant.

In a further aspect, the plant part is a plant cell or a plant tissue.In still a further aspect, the plant part is a non-regenerable cell or aregenerable cell. In another aspect the plant cell is a somatic cell. Ina further aspect the plant cell is a reproductive cell, such as an ovuleor pollen. These cells are haploid. When they are regenerated into wholeplants, they comprise the haploid genome of the starting plant. Ifchromosome doubling occurs (e.g. through chemical treatment), a doublehaploid plant can be regenerated. In one aspect the plant of theinvention is a haploid or a double haploid lettuce plant.

Moreover, there is provided an in vitro cell culture or tissue cultureof lettuce plants of the invention in which the cell- or tissue cultureis derived from a plant parts described above, such as, for example andwithout limitation, leaves, pollen, embryos, cotyledon, hypocotyls,meristematic cells, roots, root tips, anthers, flowers, seeds or stems,somatic cells, reproductive cells. For example, leaf-, hypocotyl- orstem-cuttings may be used in tissue culture.

In a specific aspect an in vitro cell culture or tissue culture oflettuce plants of the invention is provided in which the cell- or tissueculture is derived from a plant parts described above, wherein suchplant parts do not comprise reproductive cells. In another embodiment,the cell culture or tissue culture does not comprise regenerable cells.In one aspect non-propagating cells of the invention are provided and acell culture or tissue culture comprising or consisting ofnon-propagating cells of the invention.

Also provided are lettuce plants regenerated from the above-describedplant parts, or regenerated from the above-described cell or tissuecultures, said regenerated plant having Nr: 1 resistance, i.e. retainsthe introgression fragment(s) conferring Nr:1 resistance. These plantscan also be referred to as vegetative propagations of plants of theinvention.

Also provided are harvested leaves and/or heads of plants of theinvention and packages comprising a plurality of leaves and/or heads ofplants of the invention, such as 1, 2, 3, 4, 5, 10, 12, 20 heads.

The invention also provides for a food or feed product comprising orconsisting of a plant part described herein. The food or feed productmay be fresh or processed, e.g., canned, steamed, boiled, fried,blanched and/or frozen etc. Examples are salad or salad mixturescomprising leaves or parts of leaves of plants of the invention.

A lettuce plant of the invention or a progeny thereof retaining the Nr:1resistance phenotype and the introgression fragment(s), and parts of theafore-mentioned plants, can be suitably packed for, e.g., transport,and/or sold fresh. Such parts encompass any cells, tissues and organsobtainable from the seedlings or plants, such as but not limited to:heads, cuttings, pollen, leaves, parts of leaves, and the like. Headsand leaves may be harvested as baby-leaf or later. A plant, plants orparts thereof may be packed in a container (e.g., bags, cartons, cans,etc.) alone or together with other plants or materials. Parts can bestored and/or processed further. Encompassed are therefore also food orfeed products comprising one or more of such parts, such leaves or partsthereof obtainable from a plant of the invention, a progeny thereof andparts of the afore-mentioned plants. For example, containers such ascans, boxes, crates, bags, cartons, Modified Atmosphere Packagings,films (e.g. biodegradable films), etc. comprising plant parts of plants(fresh and/or processed) of the invention are also provided herein.

Plants and Progeny (Descendants)

In another embodiment, plants and parts of lettuce plants of theinvention, and progeny of lettuce plants of the invention are provided,e.g., grown from seeds, produced by sexual or vegetative reproduction,regenerated from the above-described plant parts, or regenerated fromcell or tissue culture, in which the reproduced (seed propagated orvegetatively propagated) plant comprises the Nr:1 resistance phenotype(and thus the introgression fragment(s) conferring Nr: 1 resistance).

As mentioned before, whether or not a plant, progeny or vegetativepropagation comprises the Nr:1 resistance phenotype can be testedphenotypically using e.g. the choice-test and/or non-choice test, eitherfield tests or controlled environment tests, as described above or inthe Examples, and/or using molecular techniques such as molecular markeranalysis (using one or more or all of the markers described herein), DNAsequencing (e.g. whole genome sequencing to identify the L. virosaintrogression), chromosome painting, etc.

In one embodiment, the Nr: 1 resistance QTL(s) obtainable from (obtainedfrom) NCIMB42086 or from other wild lettuces (e.g. other Nr: 1 resistantL. virosa accessions) can be combined with other genes, such as otherNr: 1 resistance genes (e.g. single genes or QTLs on differentchromosomes), with Nr:0 resistance genes (e.g. the Nr gene) or withother traits, such resistance against downy mildew, Sclerotinia rot,Botrytis, powdery mildew, anthracnose, bottom rot, corky root rot,lettuce mosaic virus, big vein, lettuce aphid, beet western yellows andaster yellows. Resistance against one or more of the following pests mayalso be present or introduced into plants of the invention: Sclerotiniaminor (leaf drop), Sclerotinia sclerotiorum (leaf drop), Rhizoctoniasolani (bottom drop), Erysiphe cichoracearum (powdery mildew), Fusariumoxysporum f. sp. lactucae (Fusarium wilt) resistance. Other resistancegenes, against pathogenic viruses (e.g. Lettuce infectious yellows virus(LIYV), lettuce mosaic virus (LMV), Cucumber mosaic virus (CMV), Beetwestern yellows virus (BWYV), Alfalfa mosaic virus (AMV)), fungi,bacteria or lettuce pests may also be introduced.

Furthermore, the invention provides for progeny (or descendants)comprising or retaining the Nr:1 resistance conferring QTL(s) of theinvention, such as progeny obtained by, e.g., selfing one or more timesand/or cross-pollinating a plant of the invention with another lettuceplant of a different variety or breeding line, or with a lettuce plantof the invention one or more times. In particular, the inventionprovides for progeny that retain QTL6.1, QTL7.1 and/or QTL7.2 as foundin NCIMB 42086. In one aspect the invention provides for a progeny plantcomprising the Nr:1 resistance, such as a progeny plant that is producedfrom a cultivated lettuce plant of the invention comprising the Nr: 1resistance by one or more methods selected from the group consisting of:selfing, crossing, mutation, double haploid production ortransformation.

Mutation may be spontaneous mutations or human induced mutations orsomaclonal mutations. See e.g. Mou 2011, Mutations in lettuceimprovement, International Journal of Plant Genomics Volume 2011,Article ID 723518.

In one embodiment, plants or seeds of the invention may also be mutated(by e.g. irradiation, chemical mutagenesis, heat treatment, TILLING,etc.) and/or mutated seeds or plants may be selected (e.g. naturalvariants, somaclonal variants, etc.) in order to change one or morecharacteristics of the plants. Similarly, plants of the invention may betransformed and regenerated, whereby one or more chimeric genes areintroduced into the plants. Transformation can be carried out usingstandard methods, such as Agrobacterium tumefaciens mediatedtransformation or biolistics, followed by selection of the transformedcells and regeneration into plants. A desired trait (e.g. genesconferring pest or disease resistance, herbicide, fungicide orinsecticide tolerance, etc.) can be introduced into the plants, orprogeny thereof, by transforming a plant of the invention or progenythereof with a transgene that confers the desired trait, wherein thetransformed plant retains the Nr:1 resistance conferringintrogression(s) and contains the desired trait.

The Nr: 1 resistance conferring QTL(s) may be transferred to progeny byfurther breeding, especially to other cultivated lettuce plants. In oneaspect progeny are F₁ progeny obtained by crossing a cultivated lettuceplant of the invention with another plant or S1 progeny obtained byselfing a plant of the invention. Also encompassed are F2 progenyobtained by selfing the F₁ plants, F3, F4 or further descendantsobtained by selfing and/or backcrossing, which retain the one or moreQTL(s). “Further breeding” encompasses traditional breeding techniques(e.g., selfing, crossing, backcrossing), marker assisted breeding,and/or mutation breeding. In one embodiment, the progeny compriseQTL6.1, QTL7.1 and/or QTL7.2 as present in NCIMB 42086.

In one aspect haploid plants and/or double haploid plants of plant ofthe invention are encompassed herein. Haploid and double haploid (DH)plants can for example be produced by anther or microspore culture andregeneration into a whole plant. For DH production chromosome doublingmay be induced using known methods, such as colchicine treatment or thelike. So, in one aspect a cultivated lettuce plant is provided,comprising one or more Nr:1 resistance conferring QTLs as describe,wherein the plant is a double haploid plant.

In another embodiment the invention relates to a method for producinglettuce seed, comprising crossing a cultivated lettuce plant of theinvention with itself or a different lettuce plant and harvesting theresulting seed. In a further embodiment the invention relates to seedproduced according to this method and/or a lettuce plant produced bygrowing such seed.

Thus, in one aspect progeny of a cultivated lettuce plant of theinvention are provided, wherein the progeny plant is produced byselfing, crossing, mutation, double haploid production or transformationand wherein the progeny retain the Nr:1 resistance described herein

In still yet another aspect, the invention provides a method ofdetermining the genotype of a plant of the invention comprisingdetecting in the genome of the plant at least a first polymorphism (e.g.one or more of the markers described herein). The method may, in certainembodiments, comprise detecting a plurality of polymorphisms in thegenome of the plant (e.g. two or more of the markers described herein,indicative of QTL6.1, QTL7.1, QTL7.2 or variants of any of these). Forexample, a sample of nucleic acid is obtained from a plant and apolymorphism or a plurality of polymorphisms is detected in said nucleicacids. The method may further comprise storing the results of the stepof detecting the plurality of polymorphisms on a computer readablemedium.

Apart from the SNP markers provided herein, also more molecular markerscan be developed by the skilled person, e.g. in-between any of themarkers provided herein, or other new markers, e.g. markers linked tovariants of QTL6.1, QTL7.1 and/or QTL7.2. The skilled person knows howto develop molecular markers. For example, this can be done by crossinga Nr:1 resistant lettuce plant of the invention (either a cultivatedlettuce or a wild lettuce, such as NCIMB42086) with a Nr:1 susceptiblelettuce plant and developing a segregating population (e.g. F2 orbackcross population) from that cross. The segregating population canthen be phenotyped for Nr:1 resistance and genotyped using e.g.molecular markers such as SNPs (Single Nucleotide Polymorphisms), AFLPs(Amplified Fragment Length Polymorphisms; see, e.g., EP 534 858), orothers, and by software analysis molecular markers which co-segregatewith the Nr: 1 resistance phenotype in the segregating population can beidentified.

In one aspect the wild L. virosa accession from which any of QTLs QTL6.1or variant, QTL7.1 or variant and/or QTL7.2 or variant are introgressedinto cultivated lettuce are not the following accessions: CGN13361,CGN16266, CGN16272, CGN04757, CGN04930, CGN04973, CGN16274, CGN21399 orCGN05148.

Deposit Information

The QTLs according to the invention were identified in, and derivedfrom, progeny of a sample of a wild L. virosa accession, PI273597(source US NGRP; National Genetic Resource Program, www.ars-grin.gov),originating from Germany (Baden Württemberg). A Nr:1 resistant plantidentified in the Examples below was grown and multiplied, and arepresentative sample of seeds were deposited by Nunhems B.V. on 5 Dec.2012 under accession number NCIMB 42086. Thus, a total of 2500 seeds ofL. virosa NCIMB 42086 were deposited by Nunhems B.V. on 5 Dec. 2012, atthe NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn,Aberdeen AB21 9YA, United Kingdom (NCIMB). Access to the deposit will beavailable during the pendency of this application to persons determinedby the Director of the U.S. Patent Office to be entitled thereto uponrequest. Subject to 37 C.F.R. § 1.808(b), all restrictions imposed bythe depositor on the availability to the public of the depositedmaterial will be irrevocably removed upon the granting of the patent.The deposit will be maintained for a period of 30 years, or 5 yearsafter the most recent request or for the enforceable life of the patentwhichever is longer, and will be replaced if it ever becomes nonviableduring that period. Applicant does not waive any rights granted underthis patent on this application or under the Plant Variety ProtectionAct (7 USC 2321 et seq.).

Various modifications and variations of the described products andmethods of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in plant breeding,chemistry, biology, plant pathology or related fields are intended to bewithin the scope of the following claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1—Nr: 1 Resistance in the Free Choice Test

1.1 Plant Material

Seeds were sown in plastic trays (4×7 pots). The plastic trays werefilled with a soil mixture composed of two different sources of peat.Once sown, the trays are placed in plastic boxes. These boxes were thenplaced on the shelves in climate cells (16/8 hours day/night cycle usingfluorescent light tubes; light intensity was approximately 50μmol·m⁻²·s⁻¹ PAR (Photosynthetically Active Radiation); temperature forday/night periods was 20/16° C.; relative humidity was set to 80%constant). During sowing, one to two seeds were placed in each pot.Approximately one week after germination, the germinated plantlets werethinned as necessary, i.e., only one plantlet was kept per pot. Theplants were kept in the climate cells (environmental conditionsdescribed above are kept throughout the whole trial) until the end ofthe experiment. Watering was done when required.

1.2 Multiplier Plant Material

Multiplier plants are the plants used to produce the aphid populationrequired for the test. The multiplier plants were sown in week 1. Seedswere germinated and plants maintained in the same way as the testedplant material. The open-field butterhead variety Mafalda(Nunhems/BayerCropScience Vegetable Seeds) was used as the multiplierplant, but other Nr:0 resistant plants could equally be used.

1.3 Trial Design

The climate cell could accommodate up to six mobile shelving units(MSU). Each unit was composed of three shelves/plateaus. A maximum ofthree plastic boxes, each containing up to 28 plants, could be stored ona single shelf.

The wild accession NCIMB42086, together with 13 other wild accessions,were compared for their resistance levels against biotype Nr: 1. In theclimate cell, 36 boxes were used. Each box contained two plants of eachplant lines/accession (the positions of the plants in each box wererandomized). The 36 boxes were then placed on the lower two plateau ofthe six MSU. No known susceptible candidates were included.

1.4 Insect

One Nr: 1 aphid isolate originally came from a lettuce field in thePfalz area in Germany. The other Nr: 1 aphid isolate originally camefrom a lettuce field near Perpignan, France.

When the aphid was not needed for trial purpose, a small colony ofinsects was retained on Mafalda “maintainer” plants (5 to 10 individualplants) in the climate cell. The plants were kept in a single box.

1.5 Inoculum Production

The rearing of the aphid population used for a trial (i.e., the“inoculum”) was started in week 4 of the planning. The rearing wasstarted by dropping nymphs and adults (wingless) aphids on the top of 3week old Mafalda plants. Approximately 500 aphids were used on a boxcontaining 28 plants. The plants and aphids were stored in boxes andkept open on shelves in the climate cell using the environmentalconditions described above. Once inoculated, the aphid population wasleft to develop on the multiplier plants for three weeks, i.e., untilrequired for the inoculation of the trial.

1.6 Trial Inoculation

The trialed plants were inoculated in week 7, i.e., when the plants were3 weeks old.

Precisely 20 aphids were transferred on the top of each tested plant.This was achieved by using a paintbrush to pick up the insects from themultiplier plants and laying them onto the heart of the tested plants.Only indeterminate nymphs and adults were used. Aphids that were visiblywinged were not included.

1.7 Trial Planning

Week Events 1 Sowing multiplier plants 4 (i) Infestation of multiplierplants with wingless adults (ii) Sowing of tested plant material 5Thinning 7 Tested plants inoculation 8 Trial scoring at 1 wpi (week postinoculation) 9 Trial scoring at 2 wpi (week post inoculation) 10 Trialscoring at 3 wpi (week post inoculation) 11 Trial scoring at 4 wpi (weekpost inoculation) 12 Trial scoring at 5 wpi (week post inoculation)1.8 Result Collection and Analysis

The number of aphids present on each individual plants was counted. Thecounting was done weekly for a period of 5 weeks post inoculation (seetrial planning above).

1.9 Results of the Free Choice Test

The results of the (free) choice-test are shown in FIGS. 2A and 2B. Theresults show that with both the German (FIG. 2A) and the French (FIG.2B) Nr: 1 isolates plants of NCIMB 42086 had very few aphids and thus ahigher level of Nr: 1 resistance. Average aphid numbers on NCIMB 42086initially increased, but only to an average number of 22 (Germanisolate) and 22.5 (French isolate). After the initial increase in aphidnumbers, numbers decreased steadily until they reached an average of1.95 (German isolate) and 2.79 (French isolate) after 5 weeks on NCIMB42086.

Thus in young plants of about 8 weeks old virtually no Nr: 1 aphids werefound on NCIMB42086 under free-choice conditions.

Example 2—Nr:1 Resistance in a Non-Choice Test

2.1 Protocol Description of the Nr: 1 Non-Choice Test

The protocol used during non-choice tests was almost identical to theone of the choice test in Example 1. Still, differences were present inthe setup of the tested plant material, in the climate cell and thedesign of the trial itself.

Contrary to the choice test, in the non-choice test the boxes wereindividually contained in plastic tents.

Additionally, a single plant genotype (single lines/accessions) was usedin each box/cage unit: in other words, no boxes contained combinationsof tested plant lines.

The resistance levels present in NCIMB 42086 and three other accessionswere compared in a non-choice test. Variety Mafalda was used as anegative control.

For each plant line, four cages were used. A box containing 18 plants ofa single line was placed in the cages. The cages were then placed in aclimate cell in a semi-randomized manner. A total of 20 cages and 360plants were used (72 plants per line or accession).

Aphid inoculation and collection of results was done as described inExample 1, except that only the German Nr: 1 isolate was tested.

2.2 Results of the Non-Choice Test

Results of the non-choice test are shown in FIG. 1A and FIG. 1B, whichshows a larger scale.

As can be seen, NCIMB 42086 has resistance against biotype Nr: 1, i.e.the average number of aphids is significantly lower than in thesusceptible control variety Mafalda. Initially the average aphid numberincreased slightly but not to above 30 (in NCIMB 42086 it was 28 aftertwo weeks), after which it decreased continuously to zero.

Thus, in young plants of about 8 weeks old no Nr: 1 aphids were found onNCIMB 42086 under non-choice conditions.

Example 2—Field Tests of Semi-Adult and Adult Plants (Free Choice andNon-Choice)

2.1 Free-Choice Field Trial

2.1.1 Plant Growth

For each trial, seeds were sown in individual mixed compost/peat “plugs”and germinated in unheated greenhouses. Approximately 7 to 9 weeks aftersowing, germinated plantlets were transplanted onto raised beds in thefield. The field was located in the province of Murcia, Spain. Wateringwas done using watering tubing placed on the top of the raised beds,amongst the rows of plants. Plants were kept in this location until theend of the trials.

2.1.2 Trial Design

Two independent trials were run during two consecutive years, referredto as Year 1 and Year 2 (Y₁ and Y₂ respectively). The same trial designwas used for both trials and his detailed below.

Five different plant lines were tested in the field, amongst them NCIMB42086 and the control varieties Mafalda and Scala (both varieties ofNunhems/Bayer CropScience Vegetable Seeds). Both Mafalda and Scala areNr:0 resistant, but Nr: 1 susceptible. Scala is a cos/Romaine type.

Raised beds were used for each trial. On each raised beds, plants weresown in replicates of 16 plants and 5 replicates were sown per raisedbed. The test was partially randomized, i.e., the trials weretransplanted to ensure that each of the plant lines were represented oneach raised bed but for each raised bed, the order of the replicates wasrandomized.

2.1.3 Insect and Inoculation

The purpose of the trials was to investigate N. ribisnigri Nr:1resistance in field conditions. The plants were not purposefullyinoculated, but left to be infested with naturally occurring populationof Nr:1 aphids. Samples of insects were collected at the end of thetrials for identification under microscopes.

2.1.4 Result Collection and Analysis

The resistance of the different plant lines tested was assessed bycounting the number of aphids present on each individual plants. Thecounting was done 14 weeks after transplanting during Trial Y₁ and 11weeks after transplanting during Trial Y₂

2.1.5—Results of the Free Choice Field Test Year 1 and Year 2

In both trials adult plants (18 to 20 weeks old) of NCIMB42086 had noaphids at all, while the control variety Mafalda and the Nr:1susceptible variety Scala both had significant numbers of aphids ofbiotype Nr:1.

2.2 Non-Choice Field Trial

2.2.1—Plant Growth

During Year 2 a non-choice, cage trial was conducted in the field,alongside the free choice trial described above. As described above,plants were germinated in unheated greenhouse before being transferredonto raised beds in the fields about 7 to 9 weeks after sowing. Waterwas also provided via tubing placed amongst the plants on the top of theraised beds. However, in contrast to the free choice trials, plants wereenclosed in cages (approximately 10 m³ each) constructed withinsect-proof mesh (size of the mesh was sufficient to prevent Thripsentry).

2.2.2—Trial Design

Two individual cages were used in the field. In each cage two raisedbeds were used. 20 plants were used on each bed, i.e., 40 plants percage.

A single plant line was transplanted in each cage, i.e., eitherNCIMB42086 or Mafalda.

2.2.3 Insect and Inoculation

N. ribisnigri aphids needed for the inoculation of the tested plantmaterial were produced on plots of Mafalda plants growing in a nearbyfield. The plots were passively infested with a naturally occurringpopulation of Nr: 1 aphids.

On the day of inoculation (plants were about 8 week old), apterousadults were collected from the Mafalda plants. The collected aphids werethen transferred individually on the top of each tested plant with thehelp of a paintbrush. Precisely 10 insects were deposited onto eachtested plant.

2.2.4—Data Collection and Analysis

The resistance of the different plant lines tested was assessed bycounting the number of aphids present on each individual plant. Thecounting was done approximately 3 weeks after inoculating.

2.2.5 Results of Non-Choice Field Trial of Semi-Adult Plants (about 11Week Old Plants)

The results are shown in FIG. 4. NCIMB42086 had no aphids at all, whilethe control variety Mafalda had significant numbers of aphids of biotypeNr: 1 (>300).

2.2.6 Conclusions of Free Choice and Non-Choice Field Trials ofSemi-Adult and Adult Plants

The results show that the Nr: 1 resistance found on young plants(Example 1, approximately 8 week old plants) of NCIMB 42086 in climatecells is highly effective in the field, both in free-choice and innon-choice trials of semi-adult and adult plants. Resistance in thefield appears to be complete (no aphids at all), both under free-choiceand non-choice conditions. NCIMB42086 is thus resistant againstdifferent Nr: 1 populations, German, French and Spanish.

Example 3—QTL Mapping of Nr:1 Resistance

In order to identify how many genetic loci and which genetic loci areresponsible for the Nr:1 resistance of NCIMB42086, NCIMB42086 wascrossed to a N. ribisnigri susceptible lettuce cultivar. The F1 wasbackcrossed to the L. sativa parent, to generate a BC1 population, whichwas used for QTL mapping.

Phenotyping data (total number of aphids of biotype Nr:1 per plant) wascollected at two time-points post inoculation, two weeks and three weekspost inoculation with aphids of biotype Nr: 1.

Results

Three QTLs were identified, one on chromosome 6 and two on chromosome 7,as shown below and on FIG. 3. The physical position on the chromosomesis based on the published lettuce genome, lgr.genomecenter.ucdavis.edu.

TABLE 1 Chromosome 6-SNP markers linked to QTL6.1 Physical positionGenotype of Genotype of SNP Genotype of NCIMB42086 heterozygous(in bases) Nr:1 suscept- (Nr:1  (Nr:1  on ible L. sativa resistantresistant Marker Position chromosome parent (wild genotype- genotypeGenomic secquence name in cM 6 type genotype) homozygous heterozygouscomprising SNP SNP_01  38.3 60688939 TT AA AT TCATATGGATTAACCATTGGTGCAAACATAGCTGCCCCTGTATA TAATCATCATCCATAAATCATT AAAT[A/T]TGTGAAGTTTTTATAAAGGTTTAGATTGTGAACAGTA AAGTTACCTGCGATTTTAGAAG GTATGTGCTTT (SEQ ID NO: 1)SNP_02 115.1 117931305 TT CC CT AATCAACATCAAGCCTCTTCAAGCTAGCTTCACAACAAGCCCTC ACGTACTCAGGTTCCCCGTGGA CTTC[C/T]GATTGACCGATTGTTTCCCCAAATTTGATCCCAAATT TCGTGGCTAATTGAACATCCTC TCTCTTCACTC (SEQ ID NO: 2)SNP_03 188.5 161579227 CC AA AC TAGGGTTTGCGAACAAGATCGAGTTGCCGGAGATTCTCCAAGGA CTGCTCTTGGCATCTTCCGACG ACAG[A/C]GGTCTTGCTCTCACTGCGACGTTGATTCTCTCCATGG TTGCTCGGATTGGAGTAGGTGG AGAGAGGGTTT (SEQ ID NO: 3)SNP_04 195.4 191681086 AA GG GA TAGAATAGATTTATTGATATGTTCCTTAATGTTGGCTTCCAAAT GTTAATCATAAGTTGTACCAAT ATGT[A/G]ATTAAATAAGTTTTAATTTAAATGCATTGAAAGGTGA AAATTATACTGTAACAAGTTTG TGAATCTTCAA (SEQ ID NO: 4)SNP_05 213.9 208949963 CC TT TC CACGACTTTGTGCAACAAAAAACATTTTAACAGTAAATGGGCAA TTTCCAGGACCAACGTTGTATG TTCA[C/T]GAAGGAGATACAATTTATGTCAAGGTCCATAACAAT GGAAGATACAACATCACCATTC ATTGGTATGAAA(SEQ ID NO: 5) SNP_06 224.7 233786307 AA CC CA TTCTTAATTTGTCTGGGACATGATGACATGTTCTGATCTTTGTCTT TTGACCTGTAGTCAACGGTCGA CC[A/C]CAACTACCACATGCGTTTGTTCTTAATCTTTTTCTGATCT GGTTTTTGTGTCGTTTTTTTTTTC CTGAAG (SEQ ID NO: 6)SNP_07 225.6 240318733 TT GG GT CGGAGCTACACGGTGTCGTTCTACTATCTCAGCGTCAGCCCTCA GGAGTCAGGTCAGACCAACATC GCGC[G/T]TTTCTTCCCAAAAACATTCTTTCCTCGAAAGCCGCAA TCGATTAGGGATTCGCTCTGCT GTTTCTTCCTT (SEQ ID NO: 7)

TABLE 2 Chromosome 6-SNP markers linked to QTL6.1 Physical positionGenotype of Genotype of SNP Genotype of NCIMB42086 heterozygous(in bases) Nr:1 suscept- (Nr:1  (Nr:1  on ible L. sativa resistantresistant Marker Position chromosome parent (wild genotype- genotypeSequence name in cM 7 type genotype) homozygous heterozygouscomprising SNP SNP_08  94.2 72772104 CC TT TC ATGTAAACTGAACCAAAAAGGCTAATCTTCCGCTTAACAACTG TAAAGCTTCTAGTGTAAATAAA GATAC[C/T]AACCCCAATTTCTCCTGATTCCATTGTGCTTAGCTCG AAATCAACCAATTGCAAACTCA ACAATCTATCA(SEQ ID NO: 8) SNP_09  83.3 93428084 TT CC CT TATATATTAAAATCAAAAAAGTTATTGATTTGATATAGTTATTTG TTTTGGCTTTAAAGTACGTACA AAA[C/T]CAATTCATTGCCAATCAAAGACAATGGTGTTCTGGTTT TCATACTAGTGGGCACATACGA CTGCATGGAG (SEQ ID NO: 9)SNP_10  77.6 107614854 GG AA AG ATTTGGTACCAAAACTTTACAATTTATACATTTACAAATTGAAG AAACCTGCGTGTTGCATCAATT GATA[A/G]GAATTGGTAACAAATTGAGCCATTTGTTTTATCTGCA TAACTCGGTAAAAACTTCTCTT GTTTGCATAAA(SEQ ID NO: 10) SNP_11  75.7 109323191 AA CC CA TCACCTCTGAAAGAAATTCAGCTTCTCCTTGTTGTGATTTGTCAA GAGCCAATTTCTTTATAGCAAC TAG[A/C]AGCCCATCTTCTAATTTACCCTATTAAAAGCCCTTAAA AGTCATATATATCTCTCTACCTT GAACAGCGT (SEQ ID NO: 11)SNP_12  48.4 unknown TT CC CT GTATATATATATTATAACCCAGACAAGTCTATTACAGCACCTCA ATAGAACGATAAAGACTCACAT AGTC[C/T]GTAAATGTTTCTCCCACAATGCCTCCACCATCTTCCT CTCGTTCCACTTCAATTGCTACC TGAATAGGTTT(SEQ ID NO: 12) SNP_13  48.4 unknown AA GG GA GGAGATGGAGCTGGCCCTATGCATTTCAAAACAAGCAACAAGTA TTATATAGCCTATCTCACTACC ATTTTATAACGATTCTGAATCTGAATGGGTTGATACACAAGCGTA AAAGAAGCCATTCAAGAGAAC ATT[A/G]AAATGATTATTACCTGCCGAAGGA (SEQ ID NO: 13) SNP_14  45.1 146394823 TT CC CTCTTGCTCCTTTGCAGCTTTGTTA GCTTCCGCTTGCTTTTTAGCCTT CTCTTTTTCCATCTCTCTCTTCGC[C/T]CGTTCTTTCTCTTTTGCA GCTTTATCCTCTTCCCTTTTCTTTTTTGCCTCTTCTTTTTCTTTTTC TTTAT (SEQ ID NO: 14)

TABLE 3 Chromosome 7-SNP markers linked to QTL7.1 Physical positionGenotype of Genotype of SNP Genotype of NCIMB42086 heterozygous(in bases) Nr:1 suscept- (Nr:1  (Nr:1  on ible L. sativa resistantresistant Marker Position chromosome parent (wild genotype- genotypeGenomic secquence name in cM 7 type genotype) homozygous heterozygouscomprising SNP SNP_15 41.5 170366427 AA TT TA AATTGTGAAATCTCCATAAATGTTTTAGGTGATGAAAAAAGGAT TGAGTGAGGACCCCTGATCAAA TTGG[A/T]CAAAAATCCACTCGATCTCTTTGAATATGTCAAAGA GTTTGTATGCCAAAAACCTGCA AATGGAAAATAA(SEQ ID NO: 15) SNP_16 33.5 196777876 AA GG GA ATTTTCGTGGTATATTTCTATCAATCAGTGCTATAAAATATCATC AGAAAATATGTGAATGATTTTA GAT[A/G]TTAAACATATATATGGTGGAGATTTTTAGTGTTAGAGG AATAGAGATAAGGTGGTATTCT AACGAAAGCG (SEQ ID NO: 16)SNP_17 29.6 208734444 CC TT TC GGGGCAAAAGTGTCCTTACGTATTGAGCACGAAAGAGAATTCTC TGTTCGCTATTGCACAAGTCTA CAGC[C/T]AGCTGTTTAAGGAACACTTTTCGGTTGAGTTGCTTTG GAGATGTTATATCCAAATCCAT TGATCCACTTG(SEQ ID NO: 17) SNP_18 29.6 212123542 CC GG GC TTGTATATTTAATCGACTTTGTAAGACTTTATTTGCCTAGCTTCA AGCTTGCTTTTTATTTATAAAAC TT[C/G]CTGTCTTTTCGATTGGTTAAATCACAAACTTTGGCTGCT TCAAGTCTTTCATTTTAATCTCT AGTCTAAC (SEQ ID NO: 18)SNP_19 27.3 218612262 AA GG GA ATGTTCATCCATGTATTACACTATTATTGTTTGTTTATGCATTGA TTTTCACTGTTTGTAGTTCTTTT AT[A/G]TCACTAGAACAATGRMATCCTTGAARGCTTTAGTGCAT CAGATCAAGCTTCAACACTCCA GTTCATTAAG (SEQ ID NO: 19)SNP_20 22.9 unknown CC TT TC CCATGWGTGTTTCTTCTTCCTCCTGCTGAAGATTTTCAGGAATTA CGCTCTCTTCAATTTTCTCTTCA TTC[C/T]TAAAAGATTCTTCATCTTCATCTTCTTCTTCCTCTTCGT TTTTCTCCRTATTTGGTTCCCAG AATCGATT (SEQ ID NO: 20)SNP_21 16.7 unknown AA CC CA CAACCACCCACMCCCAACGTTCTATCTGGCCAGATGAACT[A/C] ACGACAAGTCTTGAGATGACAG AAGAATTGATAAGAGAATAACTGTAAGACACCTCCGTTGCATTA ATAACCATGTCGGC (SEQ ID NO: 21) SNP_22 13.8232806419 TT CC CT CAGAGAGGGGTGTTTTGCTTCT TGAAAATGTCAGATTCTATAAGGAGGAAGAAAAGAACGATCCT GAATT[C/T]GCAAAGAAGCTTG CATCACTAGCAGACCTGTATGTTAATGATGCATTTGGCACTGCA CATAGAGCACATG (SEQ ID NO: 22)

Example 4—QTL Mapping of Nr: 1 Resistance

New phenotyping and marker analysis was carried out in two backcrosspopulations to map Nr:1 resistance. Phenotyping was carried out asdescribed above.

Two QTLs with significant LOD score were mapped on chromosome 6 andchromosome 7, comprising QTL6.1 and QTL7.1. Results are also shown inFIG. 3B.

TABLE 4 Chromosome 6-SNP markers linked to QTL6.1 Physical Genotype ofposition Nr:1 suscept- Nr:1 Nr:1 of SNP (in ible L. sativa resistantresistant Marker bases) on parent (wild genotype genotypeGenomic secquence name chromosome 6 type genotype) (homozygous)(heterozygous) comprising SNP SNP1.23 77007835 TT CC TCCATAAATAATAATTCGCTAATA CCCCCTGCAGTGCAAACGGGA GGGGAATCCTGGATGTTCAAGCTGGAT[T/C]GAAACTCTAGAA AAAGAGGTGATGGAATACTTT AGTGAAAATKTTAACATATTGAGAAGATGATGGYACA (SEQ ID NO: 23) SNP_02 117931305 TT CC CTAATCAACATCAAGCCTCTTCAA GCTAGCTTCACAACAAGCCCTC ACGTACTCAGGTTCCCCGTGGACTTC[C/T]GATTGACCGATTGTT TCCCCAAATTTGATCCCAAATT TCGTGGCTAATTGAACATCCTCTCTCTTCACTC (SEQ ID NO: 2) SNP2.24 145870505 CC TT CTTGCATACATACCTTAGGCAATT GGTAGCTGATGTTGAATTCTCA ATTGGTTGGAACTCTAAATGCTTCCT[C/T]AAAGTTCGTAAAAG AGAAACATGAATAGAATCAAT CAATAAGSTAGGAGACTTGCTTCTAATGGATGCCA (SEQ ID NO: 24) SNP_03 161579227 CC AA ACTAGGGTTTGCGAACAAGATCG AGTTGCCGGAGATTCTCCAAG GACTGCTCTTGGCATCTTCCGACGACAG[A/C]GGTCTTGCTCTC ACTGCGACGTTGATTCTCTCCA TGGTTGCTCGGATTGGAGTAGGTGGAGAGAGGGTTT (SEQ ID NO: 3)

TABLE 5 Chromosome 7-SNP markers linked to QTL7.1 Physical Genotype ofposition Nr:1 suscept- Nr:1 Nr:1 of SNP (in ible L. sativa resistantresistant Marker bases) on parent (wild genotype genotypeGenomic secquence name chromosome 6 type genotype) (homozygous)(heterozygous) comprising SNP SNP_17 208734444 CC TT TCGGGGCAAAAGTGTCCTTAC GTATTGAGCACGAAAGAGA ATTCTCTGTTCGCTATTGCACAAGTCTACAGC[C/T]AGCT GTTTAAGGAACACTTTTCG GTTGAGTTGCTTTGGAGATGTTATATCCAAATCCATTG ATCCACTTG (SEQ ID NO: 17) SNP17.25 211928662 CC TTCT CGCATCATCGACCTCTCAT TTAATTCATTTTCTGGTGAT CTGCCACATCAGTACTTCCAGGATTGGTCAG[C/T]AAT GAAGGAGACAAAACAAAA TGCAGCATATATGCAAKCAAATGTTGATATTTTGGGGG AAARGTACATC (SEQ ID NO: 25) SNP_18 212123542 CC GGGC TTGTATATTTAATCGACTTT GTAAGACTTTATTTGCCTA GCTTCAAGCTTGCTTTTTATTTATAAAACTT[C/G]CTGTC TTTTCGATTGGTTAAATCAC AAACTTTGGCTGCTTCAAGTCTTTCATTTTAATCTCTAG TCTAAC (SEQ ID NO: 18) SNP_19 218612262 AA GG GAATGTTCATCCATGTATTAC ACTATTATTGTTTGTTTATG CATTGATTTTCACTGTTTGTAGTTCTTTTAT[A/G]TCACT AGAACAATGRMATCCTTGA ARGCTTTAGTGCATCAGATCAAGCTTCAACACTCCAGT TCATTAAG (SEQ ID NO: 19)

QTL6.1 and QTL7.1 were identified in two different Nr:1 resistant L.virosa accession types and accession specific markers, which candistinguish introgressions and origin of the introgression wereidentified. Markers VSP1 and VSP2 are identified in one accession type,represented by NCIMB42086, and markers VSP3 and VSP4 in anotheraccession type.

TABLE 6 L. virosa accession specific markers for QTL6.1 PhysicalGenotype of position Nr:1 suscept- Nr:1 Nr:1 of SNP (in ible L. sativaresistant resistant Marker bases) on parent (wild genotype genotypeGenomic secquence name chromosome 6 type genotype) (homozygous)(heterozygous) comprising SNP VSP1 79356899 TT GG GTTTCATGCTTCTCACTCCATGTGTAAGTA GCTCCTTTATGGGTAAATGGTGTCAACGGAACAACAACTGAA[G/T]AAAATCCT TAGATAAACCTTTTGTAACATCCAACTAATCCTAGGATACTACAAGTCTCAGTG GGACTTTT (SEQ ID NO: 26) VSP3 79357567 CC AAAC AAAGTGCATAGTCTTGTGAGCTTCTTC CATGAGAAGTTCTCTCATTCCTCCTACCTTGGGCACCAAAATC[C/A]TATTTTGG AATTCGTTTAATCCTTTATTGTTTGTACTAAATGCTAACATTTTGCCTAAACGTT CCACCTT (SEQ ID NO: 27)

TABLE 7 L. virosa accession specific markers for QTL7.1 PhysicalGenotype of position Nr:1 suscept- Nr:1 Nr:1 of SNP (in ible L. sativaresistant resistant Marker bases) on parent (wild genotype genotypeGenomic secquence name chromosome 6 type genotype) (homozygous)(heterozygous) comprising SNP VSP2 203852532 AA CC ACTATAAATGTGTTGCAAGAAAACTGAAT TTCAAGAAAGCAAATGTAATCACTCTTTTTTAATTATTTGTAG[A/C]ACATCGTA CTGATCATTTTGAGAAGTTCGATCAAAAGTTTATCTATTATCCCAATTTGATCAC TTTGAAA (SEQ ID NO: 28) VSP4 212050206 AAGG AG GTTCATCAATTTCCTTTAGCTCTTTATC AAGGAAATATTCTTTTTCCTCGTAGCGAAGGGCCATATGAAT[A/G]TTTCAGAT CCAATCGTTGAAGTTTGACCCATCGAAGATAATTCTCCCAACCAATTTCATGAG TGAGACCA (SEQ ID NO: 29)

Example 5—Clip-on Cane Experiment

The two wild L. virosa accessions above comprise similarly high levelsof Nr: 1 resistance, but NCIMB 42086 has an even better resistance, asdevelopment of nymphs of Nr: 1 is stopped earlier on plants of NCIMB42086 compared to the other L. virosa accession, as found in a clip-oncage experiment.

The experiment was done using 1 day old nymphs. Per plant one cagecontaining at least 3 adult aphids was placed. One day afterinoculation, all adults were removed leaving only 3 nymphs per cage.Twelve days after removal of the adult, the number of adults (survival),the number of new nymphs and skins was counted. The experiment was donein a complete randomized design with 28 replicas (1 cage/plant). Thevariables were transformed and analyzed with ANOVA followed by LSD test.

Survival and number of new nymphs was zero for both wild L. virosaaccessions, while it was 0.86 (average survival) and 17.57 (averagenumber of new nymphs) for the susceptible control. However, the wildaccessions differed in the average number of shed skins (average numberin NCIMB42086 was 0.85, while the average number in the other accessionwas 2.11, and in the susceptible control 5.85), with NCIMB42086 having asignificantly lower average number of shed skins, showing that nymphdevelopment is stopped earlier in NCIMB42086.

The invention claimed is:
 1. A Lactuca sativa plant comprising an introgression fragment from Lactuca virosa on chromosome 6 and/or on chromosome 7 which comprises a Quantitative Trait Locus (QTL) that confers resistance against Nasonovia ribisnigri biotype 1 (Nr:1), and wherein said introgression fragment on chromosome 6 comprises QTL6.1 in the region starting at 77 Mb and ending at 162 Mb of chromosome 6 and wherein said introgression fragment on chromosome 7 comprises QTL7.1 in the region starting at 203 Mb and ending at 219 Mb of chromosome 7, wherein said introgression fragment on chromosome 6 comprises at least 1 of the following markers: a) the CC or CT genotype for the Single Nucleotide Polymorphism marker SNP1.23 in SEQ ID NO: 23; b) the CC or CT genotype for the Single Nucleotide Polymorphism marker SNP 02 in SEQ ID NO: 2; c) the TT or CT genotype for the Single Nucleotide Polymorphism marker SNP2.24 in SEQ ID NO: 24; and/or d) the AA or AC genotype for the Single Nucleotide Polymorphism marker SNP 03 in SEQ ID NO: 3, and wherein the introgression fragment on chromosome 7 comprises at least 1 of the following markers: e) the TT or TC genotype for the Single Nucleotide Polymorphism marker SNP 17 in SEQ ID NO: 17; f) the TT or TC genotype for the Single Nucleotide Polymorphism marker SNP17.25 in SEQ ID NO: 25; g) the GG or GC genotype for the Single Nucleotide Polymorphism marker SNP 18 in SEQ ID NO: 18; and/or h) the GG or GA genotype for the Single Nucleotide Polymorphism marker SNP 19 in SEQ ID NO:
 19. 2. The plant according to claim 1, comprising both the introgression fragment on chromosome 6 and on chromosome
 7. 3. The plant according to claim 1, wherein said introgression fragment on chromosome 6 further comprises: i) the GG or GT genotype for the Single Nucleotide Polymorphism marker VSP1 in SEQ ID NO: 26; or j) the AA or AC genotype for the Single Nucleotide Polymorphism marker VSP3 in SEQ ID NO:
 27. 4. The plant according to claim 1, wherein said introgression fragment on chromosome 7 further comprises: k) the CC or CA genotype for the Single Nucleotide Polymorphism marker VSP2 in SEQ ID NO: 28, or l) the GG or GA genotype for the Single Nucleotide Polymorphism marker VSP4 in SEQ ID NO:
 29. 5. The plant according to claim 1, wherein the Lactuca sativa plant is loose-leaf lettuce, Romaine lettuce, Crisphead lettuce, Butterhead lettuce, Batavia lettuce or Stem lettuce.
 6. The plant according to claim 1, wherein said introgression fragment is a fragment of chromosome 6 and/or 7 as found in L. virosa accession NCIMB42086.
 7. A seed from which a plant according to claim 1 can be grown.
 8. Leaves or heads of the plant according to claim
 1. 9. A progeny plant of the lettuce plant according to claim 1, wherein said progeny plant retains the introgression fragment on chromosome 6 comprising QTL6.1 and/or chromosome 7 comprising QTL7.1.
 10. The progeny plant according to claim 9, wherein the progeny plant is produced by one or more of: selfing, crossing, mutation, double haploid production or transformation.
 11. A part of a lettuce plant according to claim 1, wherein the part is selected from the group consisting of: stems, cuttings, petioles, cotyledons, flowers, anthers, pollen, ovaries, roots, root tips, protoplasts, callus, microspores, stalks, ovules, shoots, seeds, embryos, embryo sacs, cells, buds, leaves, and meristems, and wherein said part comprises the introgression fragment on chromosome 6 comprising QTL6.1 and/or chromosome 7 comprising QTL7.1.
 12. The plant according to claim 1, wherein said introgression fragment on chromosome 6 comprises the CC or CT genotype for the Single Nucleotide Polymorphism marker SNP_02 in SEQ ID NO: 2, and the AA or AC genotype for the Single Nucleotide Polymorphism marker for VSP3 in SEQ ID NO:
 27. 13. The plant according to claim 1, wherein said introgression fragment on chromosome 7 comprises the GG or GC genotype for the Single Nucleotide Polymorphism marker for SNP_18 in SEQ ID 18, and the GG or GA genotype for the Single Nucleotide Polymorphism marker for VSP4 in SEQ ID NO:
 29. 